Lipoparticle comprising a protein and methods of making and using the same

ABSTRACT

Enveloped virus vectors are described which comprise a cellular virus receptor protein and which are capable of fusing with a cell which comprises a viral envelope protein to which the cellular virus receptor protein is cognate. Enveloped virus vectors comprising a plurality of cellular virus receptor proteins are also described. Methods for making the enveloped virus vectors are described, as are methods of using the enveloped virus vectors. The invention further relates to a lipoparticle comprising a membrane spanning protein, and the lipoparticle can be attached to a sensor surface. The invention relates to methods of producing and using the lipoparticle to, inter alia, assess protein binding interactions.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation-in-part of U.S. patentapplication Ser. No. 09/006,678, filed on Jan. 13, 1998, which claimspriority under 35 U.S.C. §119(e), to U.S. Provisional Application No.60/047,226, filed on May 20, 1997. The present application also claimspriority to U.S. Provisional Application No. 60/257,988, filed on Dec.22, 2000.

GOVERNMENT SUPPORT

[0002] This invention was made with U.S. Government support (NIH GrantsNo. CA63531, T32-AI07325, HL 07439, and R01 40880) and the U.S.Government may therefore have certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Infection of a host cell by an enveloped virus is initiated bybinding of at least one viral envelope protein to a cognate cellularvirus receptor protein on the cell surface. The viral envelope proteinbinds to the receptor and mediates fusion of the viral envelope and thehost cell membrane. The presence or absence on a cell of a cognatecellular virus receptor protein is a primary determinant of the hostrange and the tissue tropism of a virus.

[0004] Although an enveloped virus preferentially incorporates its ownviral envelope protein(s) into the envelope during virus assembly, thetropism of a number of enveloped viruses may be altered when a differentviral envelope glycoprotein is incorporated into the envelope duringvirus assembly by a process called phenotypic mixing or pseudotyping.Virus pseudotypes may be formed by co-infection of a cell by twodifferent enveloped viruses or may be generated experimentally byexpressing a viral envelope protein encoded by one virus in a cellinfected with another virus. Pseudotype formation in vivo has beenpostulated to enhance or alter the pathologic potential of an envelopedvirus.

[0005] In addition to other viral envelope proteins, enveloped virusesmay also incorporate a number of host surface proteins, includingcellular virus receptor proteins, into their envelopes (Bubbers et al.,1977, Nature 266:458-459; Lodish et al., 1980, Cell 19:161-169; Calafatet al., 1983, J. Gen. Virol. 64:1241-1253). For example, class I andclass II major histocompatibility complex proteins, ICAM-1, ICAM-2,ICAM-3, CR3, CR4, CD43, CD44, CD55, CD59, CD63 and CD71 have beenidentified in the viral envelope of human immunodeficiency virus, HIV-1(Bastiani et al., 1997, J. Virol. 71:3444-3450). Similarly, CD55 andCD59 have been identified in human cytomegalovirus virions and also inhuman T cell leukemia virus virions (Spear et al., 1995, J. Immunol.155:4376-4381). The measles cellular virus receptor, CD46, has beenreported in the envelope of HIV-1 (Montefiori et al., 1994, Virol.205:82-92). In addition, transient high level expression in culturedcells of CD4, the primary cellular receptor for HIV-1, causes CD4 topartition into the envelope of a number of viruses, includingretroviruses, herpesviruses, and rhabdoviruses (Dolter et al., 1993, J.Virol. 67:189-195; Schnell et al., 1996, Proc. Natl. Acad. Sci. USA93:11359-11365; Schubert et al., 1992, J. Virol. 66:1579-1589; Young etal., 1990, Science 250:1421-1423). A number of factors influence theefficiency of cellular protein uptake by enveloped viruses including thesurface density of the cellular protein, the location of the cellularprotein within a cellular membrane, and the structural configuration ofthe cellular protein (Young et al., 1990, Science 250:1421-1423;Suomalainen et al., 1994, J. Virol. 68:4879-4889). Although there havebeen several reports of incorporating cellular virus receptor proteinsinto viruses, the structural and functional integrity of these proteinswas not suggested, tested, used, or demonstrated previously (Montefioriet al., 1994, Virol. 205:82-92; Dolter et al., 1993, J. Virol.67:189-195; Schnell et al., 1996, Proc. Natl. Acad. Sci. USA93:11359-11365; Schubert et al., 1992, J. Virol. 66:1579-1589; Young etal., 1990, Science 250:1421-1423).

[0006] Intracellular immunization as a method of gene therapy has beenproposed as a potential treatment for AIDS (Friedman et al., 1988,Science 335:452-454; Baltimore, 1988, Science 335:395-396). It has beenproposed that the immune system of an AIDS patient may be reconstitutedwith hematopoietic stem cells that have been rendered resistant to viralinfection by the introduction of a gene or a plurality of genes whichprotect the cell against HIV infection. Numerous intracellularantagonists of HIV replication have been developed which exhibit potentantiviral properties in vitro including trans-dominant mutants (Bevec etal., 1992, Proc. Natl. Acad. Sci. USA 89:9870-9874; Malim et al., 1992,J. Exp. Med. 176:1197-1201; Bahner. et al., 1993, J. Virol.67:3199-3207; Nabel et al., 1994, Hum. Gene Ther. 5:79-92), moleculardecoys (Sullenger et al., 1990, Cell 63:601-608; Lee et al., 1992, NewBiol. 4:66-74; Lee et al., 1994, J. Virol. 68:8254-8264), intracellularantibodies (Marasco et al., 1993, Proc. Natl. Acad. Sci. USA90:7889-7893; Duan et al., 1994, Proc. Natl. Acad. Sci. USA91:5075-5079; Shaheen et al., 1996, J. Virol. 70:3392-3400; Levy-Mintzet al., 1996, J. Virol. 70:8821-8832), anti-sense RNA (Lo et al., 1992,Virology 190:176-183; Sczakiel et al., 1992, J. Virol. 66:5576-5581;Biasolo et al., 1996, J. Virol. 70:2154-2161), ribozymes (Sarver et al.,1990, Science 247:1222-1225; Yamada et al., 1994, Gene Therapy 1:38-45;Heusch et al., 1996, Virology 216:241-244), and other antagonists(Caruso et al., 1992, Proc. Natl. Acad. Sci. USA 89:182-186; Curiel etal., 1993, Hum. Gene Ther. 4:741-747; Brady et al., 1994, Proc. Natl.Acad. Sci. USA 91:365-369).

[0007] CD4 has long been known as the cell-surface protein necessary forHIV binding to and entry into cells (Dalgleish et al., 1984, Nature312:763-767; Klatzmann et al., 1984, Nature 312:767-771; Maddon et al.,1986, Cell 47:333-348). Hence, CD4 is a cellular virus receptor proteinfor HIV. Recently, chemokine receptors have been shown to also becomponents of the HIV receptor complex and are important determinants ofHIV-1 tropism (Bates, 1996, Cell 86:1-3; D'Souza et al., 1996, NatureMed. 2:12931300; Weiss, 1996, Science 272:1885-1886). HIV envelopeproteins interact with CD4 and chemokine receptors present on thesurface of a cell, resulting in binding of the virus to the cellfollowed by virus-mediated fusion of the viral envelope and the cellmembrane. Initial binding of the HIV envelope protein, gp120, to CD4triggers one or more conformational changes in one or more HIV viralenvelope glycoproteins. Subsequent interaction of an HIV envelopeprotein with a chemokine receptor on the surface of the cell causesexposure of the viral gp41 fusion peptide to the cell membrane, andultimately results in fusion of the viral envelope and cell membrane(Collman et al., 1989, J. Exp. Med. 170:1149-1163).

[0008] Chemokine receptors are seven transmembrane-spanning G-proteincoupled receptors and are divided into two classes, the CC-class and theCXC-class of chemokine receptors. These two classes of chemokinereceptors differ in their tissue distribution, their ligand specificity,and their capacity to specifically interact with particular viruses,including particular isolates of HIV and SIV. CCR5 is a chemokinereceptor in the CC-class and CXCR4 is a chemokine receptor in theCXC-class.

[0009] The ability of HIV-1 to infect T-cells is well known. T-celltropic strains of HIV-1 undergo envelope-mediated fusion with and enterinto T-cells only if the T-cells express both CXCR4 and CD4 (Berson etal., 1996, J. Virol. 70:6288-6295; Feng et al., 1996, Science272:872-877).

[0010] Macrophages and other mononuclear phagocytes are an importantreservoir for virus replication in HIV-infected individuals and aresuspected to be a major source of ongoing virus replication in patientsreceiving anti-retroviral therapy (Gartner et al., 1986, Science233:215-219; Ho et al., 1995, Nature 373:123-126; Wei et al., 1995,Nature 373:117-122; Coffin, 1995, Science 267:483-489).Macrophage-tropic strains of HIV-1 undergo envelope mediated fusion withand enter into macrophages only if the macrophages express both CCR5 andCD4 (Choe et al., 1996, Cell 85:1135-1148; Doranz et al., 1996, Cell85:1149-1158; Deng et al., 1996, Nature 381:661-666; Dragic et al.,1996, Nature 381:667-673; Alkhatib et al., 1996, Science 272:1955-1958).Similarly, SIV undergoes envelope mediated fusion with and enters intocells only if the cells express both CCR5 and CD4, although otherunidentified cellular virus receptor proteins have been also implicatedin SIV infection (Chen et al., 1997, J. Virol. 71:2705-2714; Edinger etal., 1997, Proc. Natl. Acad. Sci. USA 94:4005-4010; Marcon et al., 1997,J. Virol. 71:2522-2527). Many primary isolates of HIV-1 are capable ofundergoing envelope mediated fusion with and entering into cells whichexpress CD4 and at least one chemokine receptor, including, but notlimited to, CXCR4 and CCR5 (Choe et al., 1996, Cell 85:1135-1148; Doranzet al., 1996, Cell 85:1149-1158; Simmons et al., 1996, J. Virol.70:8355-8360; Connor et al., 1997, J. Exp. Med. 185:621-628; He et al.,1997, Nature 385:645-649). Therefore, expression of chemokine receptorsor other cellular virus receptor proteins on the surface of cellsappears to be a major determinant of enveloped viral tropism.

[0011] Young et al. have demonstrated that CD4 is efficientlyincorporated into the envelopes of retroviral particles. However, theseparticles failed to enter cells expressing HIV envelope glycoproteins(Young et al., 1990, Science 250:1421-1423). Recently, Schnell et al.reported that CD4 may be packaged into vesicular stomatitis virus(Schnell et al., 1996, Proc. Natl. Acad. Sci. USA 93:11359-11365). Thus,to date, production of virus particles comprising host cell receptors orother surface proteins while preserving the biological function of themolecule, has not been achieved.

[0012] Ligand interactions with membrane proteins are responsible for amultitude of cell adhesion, signaling, and regulatory events. Thisdiversity of functions makes membrane proteins, such as seventransmembrane domain (7TM) receptors, important drug targets. Proteinsthat span the membrane multiple times present a unique set of challengesfor ligand binding studies because they require a lipid environment tomaintain native structure. Whereas detergent conditions can occasionallybe found that allow native structure to be maintained in solution, thisis an empirical and frequently time-consuming process. As a result,ligand binding studies involving 7TM and many other membrane proteinstypically involve using whole cells or vesicles derived from cellmembranes, where the protein of interest is a minor component.

[0013] Interactions between the HIV-1 envelope (Env) protein and itsreceptors underscore both the strengths and weaknesses of cell-surfacebinding assays. HIV-1 Env mediates virus entry by sequentially bindingto CD4 and a coreceptor, with these interactions triggeringconformational changes in Env that lead to membrane fusion (Berger etal., 1999, Annu. Rev. Immunol. 17:657-700). R5 virus strains that areresponsible for virus transmission use the 7TM chemokine receptor CCR5in conjunction with CD4 to enter cells, X4 virus strains that tend toevolve years after infection use the related CXCR4 receptor, andintermediate dual-tropic R5X4 virus strains can use both receptors.Binding of the soluble gp120 subunit of Env to CD4 is readily detected,and gp120 proteins from some R5 virus strains bind to CCR5 with highaffinity (Doranz et al., 1999, J. Virol. 73:10346-10358 and Doranz etal., 1999, J. Virol. 73:10346-10358). However, direct binding of X4gp120 proteins to CXCR4 has been difficult to measure, as has binding ofR5X4 gp120 proteins to either CXCR4 or CCR5 (Doranz et al., 1999, J.Virol. 73:2752-2761, Baik et al., 1999, Virology 259:267-273 andEtemad-Moghadam et al., 2000, J. Virol. 74:4433-4440). Interactionsbetween Env and alternative coreceptors such as CCR3 and STRL33 alsocannot be measured using standard binding techniques (Baik et al., 1999,Virology 259:267-273). As virus receptor interactions can be the targetsof neutralizing antibodies and small molecule inhibitors (reviewed inref. 1), improved assays to measure these binding events are needed.

[0014] An approach that in principle would make it possible to monitorlow affinity but functionally important Env-coreceptor interactionswould be to use microfluidic devices, e.g., biosensors (optical and SPRbiosensors), and other analytical instruments that detect interactionsbetween molecules, preferably in real-time. The most commonly usedoptical biosensors (Biacore, Uppsala, Sweden) are based on surfaceplasmon resonance, which measures changes in refractive index at thesensor surface (Canziani et al., 1999, Methods 19:253-269 and Rich etal., 2000, Curr. Opin. Biotechnol. 11:54-61). With this technique, oneprotein is tethered to the biosensor surface, and changes in refractiveindex that occur upon exposure to its binding partner are monitored.However, a general method for attaching intact membrane proteins to thisinstrument does not exist. Membrane proteins can span the membranemultiple times, can form homo- or hetero-oligomers in the membrane, andremoval from the lipid bilayer can destroy tertiary or quaternarystructure. Thus, despite the importance of membrane proteins inbiological processes, to date, there is no method to study the complexinteraction between these molecules and molecules that specificallyinteract with them using powerful techniques such as, but not limitedto, using optical biosensors.

[0015] To date, there are a limited number of therapies directed againstHIV infection in humans, each having a variable success rate, generallyconcomitant with the emergence of strains of HIV which are resistant tothe therapy. There remains an acute need for the development of anti-HIVtherapies to which the virus cannot develop resistance. The presentinvention satisfies this need.

[0016] Further, there is long-felt need for assays for the study of cellmembrane protein-protein interactions, and the present invention alsosatisfies this need.

SUMMARY OF THE INVENTION

[0017] The invention includes an isolated lipoparticle comprising amultiple membrane spanning protein wherein the protein is not CD63. Inone aspect, the protein is capable of binding with a ligand underconditions wherein the ligand would bind with an otherwise identicalprotein present on a cell membrane.

[0018] In another aspect, the lipoparticle is a virus.

[0019] In yet another aspect, the virus is a membrane-enveloped virus.

[0020] In a further aspect, the membrane-enveloped virus is aretrovirus.

[0021] In an even further aspect, the virus is selected from the groupconsisting of a murine leukemia virus, a human immunodeficiency virus, arabies virus, a Rous sarcoma virus, and a vesicular stomatitis virus.

[0022] In another aspect, the protein is selected from the groupconsisting of a G-protein coupled receptor, a transporter protein, andan ion channel protein.

[0023] In one aspect, the protein is selected from the group consistingof CCR5, CXCR4,, MCAT-1, CXCR2, CXCR3, mu-opioid receptor, and KCNH2potassium channel protein.

[0024] The invention includes a composition comprising an isolatedlipoparticle attached to a sensor surface, the lipoparticle furthercomprising a membrane spanning protein.

[0025] In one aspect, the protein is selected from the group consistingof a transport protein, a G-protein coupled receptor, an ion channelprotein, a type I membrane protein, and a type II membrane protein.

[0026] In another aspect, the G-protein coupled receptor is selectedfrom the group consisting of a mu-opioid receptor, a CXCR2, CXCR3,CXCR4, a CCR5, a CCR8, a XCR1, and a CX3CR1.

[0027] In yet another aspect, the ion channel protein is selected fromthe group consisting of KCNH2 potassium channel protein, Kv1.3 potassiumchannel protein, and CFTR protein.

[0028] In a further aspect, the transporter protein is selected from agroup consisting of a glucose transporter protein and an amino acidtransporter protein.

[0029] In another aspect, the type I membrane protein is selected fromthe group consisting of CD4, Tva, and neuropilin-2.

[0030] In yet another aspect, the type II membrane protein comprisesDC-specific ICAM-3 grabbing nonintegrin (DC-SIGN).

[0031] In one aspect, the lipoparticle is a virus.

[0032] In another aspect, the virus is a membrane-enveloped virus.

[0033] In yet another aspect, the membrane-enveloped virus is aretrovirus.

[0034] In a further aspect, the virus is selected from the groupconsisting of a murine leukemia virus, a human immunodeficiency virus, arabies virus, a Rous sarcoma virus, and a vesicular stomatitis virus.

[0035] In another aspect, the lipoparticle further comprises a plasticbead core to form a proteoliposome.

[0036] In yet another aspect, the sensor comprises a microfluidicdevice.

[0037] In a further aspect, the microfluidic device is a biosensor.

[0038] In another aspect, the biosensor is an optical biosensor.

[0039] In a further aspect, the optical biosensor measures surfaceplasmon resonance (SPR).

[0040] In yet another aspect, the surface is located on a biosensorchip.

[0041] In another aspect, the biosensor chip is selected from the groupconsisting of a gold coated biosensor chip, a gold and dextran coatedbiosensor chip, and a derivatized gold biosensor chip.

[0042] The invention also includes a method of assessing the bindinginteraction of a membrane spanning protein with a ligand. The methodcomprises (a) producing a lipoparticle comprising a membrane spanningprotein; (b) attaching the lipoparticle to a substrate; (c) contactingthe protein present on the lipoparticle with a ligand of the protein;and (d) detecting any change in the substrate compared with any changein an otherwise identical substrate wherein the protein present on thelipoparticle is not contacted with the ligand, wherein detecting achange in the substrate wherein the protein present on the lipoparticleis contacted with the ligand compared with the otherwise identicalsubstrate wherein the protein present on the lipoparticle is notcontacted with the ligand assesses the binding interaction of theprotein with the ligand.

[0043] In one aspect, the detecting in (d) is performed using amicrofluidic device and the substrate is a sensor surface.

[0044] In another aspect, the microfluidic device is a biosensor device.

[0045] In yet another aspect, the biosensor device comprises amicrochannel or a microwell.

[0046] In a further aspect, the biosensor is an optical biosensor.

[0047] In yet a further aspect, the optical biosensor is a surfaceplasmon resonance biosensor device.

[0048] The invention includes a method of identifying a potential ligandof a membrane protein. The method comprises (a) attaching a lipoparticlecomprising a membrane protein to a surface; (b) contacting the proteinwith a test ligand; and (c) comparing the surface comprising thelipoparticle comprising the protein contacted with the test ligand withan otherwise identical surface comprising an otherwise identicallipoparticle comprising a protein not contacted with the test ligand,wherein a difference between the surface comprising the lipoparticlecomprising a protein contacted with the test ligand compared with theotherwise identical surface comprising the otherwise identicallipoparticle comprising the protein not contacted with the test ligandis an indication that the ligand is a potential ligand of the protein.

[0049] The invention includes a ligand identified by this method.

[0050] In one aspect, the comparing in (c) is performed using amicrofluidic device.

[0051] In another aspect, the microfluidic device is a biosensor device.

[0052] In yet another aspect, the protein is selected from a multiplemembrane spanning protein and a single membrane spanning protein.

[0053] In another aspect, the multiple membrane spanning protein isselected from the group consisting of a G-coupled protein receptor(GCPR), a transporter, and an ion channel.

[0054] In a further aspect, the single membrane spanning protein isselected from the group consisting of a type I membrane protein and atype II membrane protein.

[0055] In another aspect, the test ligand is selected from the groupconsisting of a protein and a chemical compound.

[0056] In yet another aspect, the protein is an antibody.

[0057] The invention includes a method of identifying a compound thataffects binding between a ligand and a membrane protein receptor. Themethod comprises (a) attaching a lipoparticle comprising a membraneprotein to a surface; (b) contacting the protein with a known ligandunder conditions wherein the protein specifically binds with the ligand;(c) contacting the lipoparticle of (b) with a test compound; and (d)comparing the surface comprising the lipoparticle contacted with thetest compound with an otherwise identical surface comprising anotherwise identical lipoparticle not contacted with the test compound,wherein a difference between the surface comprising the lipoparticlecontacted with the test compound compared with the otherwise identicalsurface comprising the otherwise identical lipoparticle not contactedwith the test compound is an indication that the test compound affectsbetween the ligand and the membrane protein receptor.

[0058] The invention includes a kit for assessing the bindinginteraction of a membrane spanning protein with a ligand. The kitcomprising a lipoparticle comprising a membrane spanning protein and asubstrate, the kit further comprising an applicator, and aninstructional material for the use thereof.

[0059] In one aspect, the kit further comprises a ligand of the protein.

[0060] The invention includes a kit for identifying a potential ligandof a membrane protein. The kit comprises a lipoparticle comprising amembrane protein and a surface, the kit further comprises an applicator,and an instructional material for the use thereof.

[0061] In one aspect, the kit further comprises a test ligand.

[0062] The invention also includes a kit for identifying a compound thataffects binding between a ligand and a membrane protein receptor. Thekit comprises a lipoparticle comprising a membrane protein and asurface. The kit further comprises an applicator, and an instructionalmaterial for the use thereof.

[0063] In one aspect, the kit further comprises a test compound.

[0064] In another aspect, the kit further comprises a known ligand ofthe membrane protein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0065]FIG. 1 is a pair of diagrams depicting the location of viralenvelope proteins and cellular virus receptor proteins on the surfacesof viruses and cells. In Panel A, a diagram of the envelope of a virushaving a viral envelope protein embedded in the envelope thereof isdepicted along with a diagram of a cell membrane having at least twodifferent cellular virus receptor proteins embedded therein. In Panel B,there is depicted a diagram of an enveloped virus vector of theinvention, wherein the envelope of the vector comprises two differentcellular virus receptor proteins. Also in Panel B, there is depicted adiagram of a cell membrane having a viral envelope protein embeddedtherein.

[0066]FIG. 2, comprising FIGS. 2A and 2B, is a pair of graphs depictingreporter gene activity as a function of the target cell and the viruswhich is used to deliver the reporter gene to target cells. The bargraph in FIG. 2A depicts luciferase activity measured in CEMx174 cellswhich were chronically infected with HIV or SIV and which were contactedwith the enveloped virus vector indicated at the bottom of the graph.Because each enveloped virus vector used encoded luciferase, the levelof luciferase detected corresponds to the ability of the vector to fusewith the cell. The graph in FIG. 2B depicts cell surface expression ofviral envelope glycoprotein gp120 in CEMx174 cells which were infectedwith SIVmac239 or with SIVmac239/MT or which were not infected, asassessed by FACS.

[0067]FIG. 3, comprising FIGS. 3A and 3B, is a pair of bar graphsdepicting the results of experiments in which enveloped virus vectorswere contacted with CEMx174 cells treated with α-CD4#19 antibody, whichantibody binds specifically to CD4. In FIG. 3A, the enveloped virusvector used in each experiment was pre-incubated with either α-CD4#19antibody or a control antibody prior to mixing the vector with thecells. Luciferase activity levels assessed following transfection ofcells with a vector incubated with α-CD4#19 antibody are represented bysolid bars. Luciferase activity levels assessed following transfectionof cells with a vector incubated with a control antibody are representedby striped bars. In FIG. 3B, the enveloped virus vector used in eachexperiment was pre-incubated with one of two antibodies as in Panel A(striped bars), or the vector was not preincubated with an antibody andone of the two antibodies was added sixteen hours after the vector andcells were mixed (solid bars).

[0068]FIG. 4 is a graph depicting the level of luciferase activitymeasured following contact of non-infected and HIV-infectedmonocyte-derived macrophages (MDM) with enveloped virus vectors. Stripedbars represent results obtained using non-infected MDM. Solid barsrepresent results obtained using HIV-1/89.6-infected MDM.

[0069]FIG. 5, comprising FIGS. 5A and 5B, is a pair of images depictingthe results of Western blot analysis of proteins in fractions obtainedfrom a density gradient sedimentation centrifuge tube. Virions obtainedduring preparation of MLV(Tva) were layered onto the solution in thecentrifuge tube prior to centrifugation. Each lane in the imagescorresponds to a single density gradient fraction, and the densitycorresponding to the fraction is indicated at the top of each lane. InFIG. 5A, an antibody which specifically binds to MLV Gag protein wasused to visualize Gag protein. In FIG. 5B, an antibody whichspecifically binds to Tva protein was used to visualize Tva protein.

[0070]FIG. 6, comprising FIGS. 6A and 6B, is a pair of graphs depictingthe inhibitory effects of a protein on the ability of MLV(Tva) to fusewith 3T3EnvA cells. The graph in FIG. 6A depicts results obtained usinganti-Tva, an antibody which specifically binds to Tva and whichinterferes with the interaction between Tva and EnvA. The graph in FIG.6B depicts the results using sTva, a soluble form of Tva. “% inhibition”refers to the decrease in the number of cells which fused with theenveloped virus vector, which decrease was observed in the presence ofthe indicated concentration of the indicated compound, relative to whenthe compound was not present.

[0071]FIG. 7, comprising Panels A and B, is a pair of diagrams whichdepict infection of cells by enveloped viruses and assembly and buddingof enveloped viruses. In Panel A, a normal cycle of cellular infectionis depicted. In Panel B, a means of altering the tropism of an envelopedvirus is depicted.

[0072]FIG. 8, comprising FIGS. 8A and 8B, is an image of Western blotsof MLV pseudotypes. FIG. 8A is an image of a Western blot demonstratingthat MLV particles were produced by cotransfection of 293T cells withplasmids expressing MLV gag and either the indicated receptor constructsor an empty pCDNA3vector (MLV-pCDNA3). Purified MLV-pCDNA3, MLV-CCR5,MLV-CXCR4, and MLV-CD4 particles were analyzed by Western blot usingantibodies against the various receptors and the MLV gag protein asindicated. FIG. 8B is an image depicting a Western blot where fractionsfrom an equilibrium density gradient containing MLV-CCR5 were analyzedby SDS-PAGE and Western blot using antibodies to CCR5 (Upper panel) andMLV-gag (Lower panel). Densities of each fraction are indicated (ing/ml), and a CCR5 standard was run in the far right lane to control forexpression.

[0073]FIG. 9, comprising FIGS. 9A through 9F, are graphs depictingantibody binding to chemokine receptor pseudotypes. FIG. 9A depictsequivalent amounts of MLV-CXCR4 and MLV-pCDNA3 were attached to aBiacore F1 chip. Binding of 333 nM 12G5 (anti-CXCR4 antibody) and 666 nMCTC8 (anti-CCR5 antibody) to MLV-CXCR4 and MLV-pCDNA3 is demonstrated.Binding was measured for 120 seconds before washing with PBS runningbuffer for an additional 120 seconds to measure dissociation.Regeneration pulses are indicated by bars. Instrument noise between theregeneration pulses is due to changes in flowrate and the injections ofthe regeneration buffer, which lead to immediate shifts in the signalbaseline. The slower changes in signal reflect binding of proteins tothe sensor surface. FIG. 9B depicts an assay wherein equivalent amountsof MLV-CCR5 and MLV-pCDNA3 were attached to a Biacore F1 chip, and thebinding of 400 nM CTC8 or 800 nM 12G5 to MLV-CCR5 and MLV-pCDNA3isshown. A single regeneration pulse (bar) was used to strip boundantibody. FIG. 9C depicts data from six sequential injections of 166 nM12G5 to MLV-pCDNA3 and MLV-CXCR4 are overlayed. In all cases, and in allsubsequent figures, the sensorgrams show subtracted data, in which thesignal obtained from the control surface is subtracted from the signalobtained from the surface bearing receptor-positive particles. Bindingwas measured for 60 seconds. Regeneration conditions were similar tothose used in FIG. 9A.

[0074]FIG. 9D depicts subtracted data from the binding of 5 nM12G5 toMLV-CXCR4 and MLV-pCDNA3 which is shown in green. After regeneration,binding in the presence of the CXCR4 inhibitor ALX40-4C (at 4 mM) isshown in red, whereas binding of the antibody following washout of theinhibitor is shown in blue. FIG. 9E Subtracted data for serialinjections of 111 nM 12G5 to MLV-CXCR4 and MLV-pCDNA3 at different flowrates are shown. Regeneration conditions were similar to those used inA. FIG. 9F Subtracted data from binding of serial dilutions of CTC8 toMLV-CCR5 and MLV-pCDNA3 are shown.

[0075]FIG. 10, is a graph depicting bivalent binding of 12G5 toMLV-CXCR4. Sensorgrams of 12G5 binding to MLV-CXCR4 are shown atdifferent mAb concentrations, with the signals obtained from 12G5binding to the MLV-pCDNA3 surface being subtracted. Binding was measuredfor 90 s and dissociation for 80 s before regeneration. Global analysisof the data using Biaevaluation 3.0 software was performed, and theboxes indicate the best fit of the data to a bimolecular interaction.

[0076]FIG. 11, comprising FIGS. 11A-11C, is a graph depicting HIV-1 gp120 binding to MLV-CXCR4. FIG. 11A Equivalent amounts of MLV-pCDNA3 andMLV-CXCR4 were attached to a Biacore C1 chip, and the binding of 400 nM8x gp 120 was measured to both surfaces in a running buffer of DMEM with0.1% Pluronic F127. Binding was measured for 120 seconds anddissociation for 300 seconds. Two brief regeneration pulses with pH9/NaCl were used to strip gp 120 from the surface. The signal from thepCDNA3 control surface was subtracted in B and C. FIG. 11B is an imagedemonstrating the ability of mAb 17b, which binds to the conservedcoreceptor binding site in Env, to block 8x binding to CXCR4 wasmeasured. Subtracted data for the association phase of 150 nM 8x toMLV-CXCR4 and MLV-pCDNA3 are shown (8x). Association of 750 nM 17b aloneand 150 nM 8x prebound to 750 nM 17b is also shown. These experimentswere performed using a Biacore F1 chip in PBS running buffer.Regeneration was achieved previously herein in FIG. 8A, supra. FIG. 11C,this is an image depicting subtracted data for binding of serialdilutions of 8x gp120 to MLV-CXCR4 and MLV-pCDNA3.

[0077]FIG. 12, comprising FIGS. 12A and 12B, depicts binding ofcollapsin-1 to MLV-NP-1 pseudotypes. FIG. 12A is an image of a westernblot depicting MLV-NP-1, MLV-P1x-2, MLV-CCR5, and MLV-CXCR4 preparationsblotted with an antibody against the myc epitope, which was present onthe C terminus of the NP-1 and P1x-2 constructs. Equivalent amounts ofMLV gag were present in these samples. FIG. 12B is a graph depictingequivalent amounts of MLV-CCR5 and MLV-NP-1 attached to a Biacore C 1chip, and binding of 200 nM collapsin-1 was measured to both surfaces inPBS running buffer. The sensorgram shows the MLV-CCR5 signal subtractedfrom the MLV-NP-1 surface. Collapsin-l was injected for 150 seconds, andthe arrow indicates the beginning of the wash step. A brief pulse with 2M MgCl₂ was sufficient to regenerate the surface following binding.

DETAILED DESCRIPTION OF THE INVENTION

[0078] The invention relates to an enveloped virus vector whichcomprises a cellular virus receptor protein and which is capable offusing with a cell which comprises a viral envelope protein to which thecellular virus receptor protein is cognate. In certain embodiments, theenveloped virus vector comprises a plurality of cellular virus receptorproteins.

[0079] The invention also relates to methods of making the envelopedvirus vector of the invention including, but not limited to, a methodinvolving a producer cell which comprises a cellular virus receptorprotein and a method involving a producer cell which does not normallycomprise a cellular virus receptor protein.

[0080] The invention further relates to methods of using the envelopedvirus vector of the invention including, but not limited to, a method ofdirecting delivery of a composition to a cell, particularly to anenveloped virus-infected cell, a method of altering a property such asthe host range or tissue tropism of an enveloped virus, a method ofrendering a cell susceptible to fusion with an enveloped virus or anenveloped virus vector, a method of producing a cellular virus receptorprotein, a method of identifying a viral envelope protein, a method ofidentifying a cellular virus receptor protein, and a method ofidentifying a composition capable of affecting the interaction between aviral envelope protein and a cellular virus receptor protein which iscognate thereto.

[0081] The invention also relates to methods of using the envelopedvirus of the invention to assess protein-protein binding interactionsusing methods such as, but not limited to, microfluidics-based assays.The enveloped virus vector, also referred to herein as a lipoparticle,allows presentation of a cell membrane protein while preserving itsbiological function, such that the interaction of the protein with itscognate ligand can be studied.

[0082] The Enveloped Virus Vector of the Invention

[0083] Infection of a host cell by an enveloped virus involves theinteraction of at least one virus-encoded viral envelope protein, whichis located on the outer surface of the viral envelope, with at least onecognate cell-encoded cellular virus receptor protein, which isassociated with the outer surface of the host cell outer membrane. Theinvention relates to the discovery that an enveloped virus vectorcomprising a cognate cellular virus receptor protein is capable offusing with a cell comprising a viral envelope protein.

[0084] The enveloped virus vector of the invention is a virus-likeparticle which comprises a protein, wherein the biological activity orfunction, conformation, or both, of the protein are retained comparedwith the activity, function or conformation of the protein present inits native state in a membrane. For instance, where the protein is acellular virus receptor protein, the enveloped virus vector is capableof binding to and fusing with a target cell which comprises a viralenvelope protein to which the cellular virus receptor protein iscognate.

[0085] The ability of the cellular virus receptor protein to interactwith molecules in the solvent in which the vector is suspended, when thecellular virus receptor protein is a component of the vector, isanalogous to the ability of the cellular virus receptor protein tointeract with molecules in the extracellular solvent when the cellularvirus receptor protein is expressed on the surface of a cell. Likewise,the ability of the membrane protein of the lipoparticle is analogous tothe ability of the membrane protein to interact with a molecule when themembrane protein is expressed on the surface of a cell. This is so, evenwhere the lipoparticle is attached to a support, such as, but notlimited to, a sensor chip.

[0086] Moreover, the enveloped virus vector, also referred to herein asa lipoparticle, comprises, in essence, an exterior lipid bilayercomprising a membrane protein. Thus, the lipoparticles of the inventioncomprise a simple membrane in which a protein of interest can beembedded while maintaining the normal structure, function, or both, ofthe protein. That is, the membrane protein may not retain a detectablefunction in a lipoparticle since this is partly determined byintracellular pathways that may or may not be present inside thelipoparticle. Preferably, however, the membrane protein maintains itsstructure compared with the native protein when present in the membrane.As exemplified herein, the membrane protein when present in alipoparticle can mediate virus fusion thereby also maintaining itsfunction as mediating membrane fusion because no intracellular proteinsare needed for fusion. The same would not be the case for, e.g., GCPRsignaling, because intracellular G-proteins and downstream pathways maybe necessary for the protein to function as it does when present in itsnative form in the membrane. A “lipoparticle,” as that term is usedherein, means a small particle of about a nanometer to about onemicrometer, comprising a lipid bilayer further comprising a proteinwhere the protein can interact with a cognate ligand essentially as itwould otherwise interact with the ligand when the protein is present inan intact membrane. The lipoparticle does not encompass cell membranevesicles, which are typically produced using empirical methods and whichare usually heterogeneous in size. The lipoparticle of the invention is,preferably, dense, spherical and/or homogeneous in size.

[0087] The data disclosed herein demonstrate, for the first time, thatcomplex cell membrane proteins, which can, but need not, span the lipidbilayer many times, can be presented in the context of a relativelysimple lipid bilayer of, for example, a virus vector particle and yet,surprisingly, the protein retains its biological structure, function, orboth. That is, the data disclosed herein demonstrate that seventransmembrane domain proteins, such as chemokine receptors, can beembedded in a virus vector, i.e., a lipoparticle, and can mediate fusionof the virus vector with a cell where the cell expresses a cognatebinding partner that binds with the transmembrane protein. Thisdiscovery makes it possible not only to use the virus vector to delivera substance of interest to a cell expressing any target protein forwhich a cognate receptor is known, but also makes possible the study ofcomplex protein-protein interactions between a membrane protein and itscognate ligand(s).

[0088] The lipoparticle allows the stable presentation of structurallyintact complex membrane proteins within a particulate format that issuitable for gene delivery, microfluidics, biosensors, and antigenpresentation. That is, because the structure of complex membraneproteins can be maintained using the lipoparticle, the present inventionincludes methods of using lipoparticles comprising a membrane protein ofinterest as an immunogenic vector for production of antibodies thatspecifically bind with the membrane protein. The antibodies produced bythis method can bind with the protein in its native structure and thuscan provide a method for producing antibodies that can, for instance,inhibit protein function by steric blocking of important sites on theprotein and/or antibodies that can affect protein function by allostericeffect.

[0089] The enveloped virus vector of the invention can comprise one morecellular virus receptor proteins. Although the Examples described hereindisclose enveloped virus vectors which comprise one or two cellularvirus receptor proteins, one skilled in the art is enabled by theteaching provided herein to produce an enveloped virus vector comprisingany number of cellular virus receptor proteins in the envelope thereof.Indeed, one skilled in the art, based on the disclosure provided herein,would appreciate that the invention encompasses any membrane protein andany protein typically present in a membrane can be inserted into thelipoparticles of the invention thereby presenting the protein in itsnative conformation and/or preserving it's binding affinity of a cognateligand or binding partner.

[0090] Indeed, the data disclosed herein demonstrate that, surprisingly,complex cell membrane protein can, within the context of a simplevirally derived lipid bilayer, can form complexes and can interact suchthat function of the protein is conserved. More specifically, the datademonstrate that CD4 and a chemokine receptor present on thelipoparticle can interact and bind with the viral env when the cellmembrane proteins are present on a viral membrane. These unexpectedresults demonstrate that the lipoparticle can comprise multiple membraneproteins wherein the proteins can form homo- and/or heterodimers, orotherwise interact, substantially as they would otherwise do in thecontext of the membrane where they are typically found. Thus, thepresent invention includes a lipoparticle comprising at least onemultiple membrane spanning protein and can further comprise additionalmembrane components that can interact with the protein in a mannersubstantially similar to the interaction of these components in anintact membrane.

[0091] It is known in the art that enveloped virus particles can beproduced which are missing one or more of the ordinary components ofsuch particles, such as a portion of the genome of the enveloped virus(Volt et al., 1977, Annu. Rev. Genet. 11:203-238; Hanafusa, 1977, In:Comprehensive Virology, vol. 10, Fraenkel-Conrat et al., eds., PlenumPress, New York, pp. 401-483). Such virus particles are referred toherein as ‘defective.’ Enveloped virus vectors comprising such adefective virus particle and a cellular virus receptor protein areincluded in the present invention. It is contemplated that the omissionof one or more components of such particles provides an opportunity tosubstitute an additional component in place of the missing component. Inaddition, numerous viruses known in the art that are able to accommodatethe presence of an additional component without deletion of a componentof the virus. By way of example, the additional component may be anucleic acid, an antisense nucleic acid, a gene, a protein, a peptide,Vpr protein, an enzyme, an intracellular antagonist of HIV, aradionuclide, a cytotoxic compound, an antiviral agent, an imagingagent, and the like.

[0092] The enveloped virus vector of the invention may compriseadditional components beyond those specifically recited herein. Thesuitability of the enveloped virus vector of the invention forspecifically targeting cells of one or more particular phenotypesrenders the vector an appropriate vehicle for delivering theseadditional components to such cells, the additional component being anyone or combination of those recited herein.

[0093] Similarly, the lipoparticles of the invention can be used toassess the binding of the protein presented in the lipid bilayer of theparticle with a test component and or to assess the effect of a testcompound on the binding of the protein with a cognate ligand. This isbecause, as more fully set forth elsewhere herein, the protein embeddedin the lipoparticle retains its ability to bind with its cognateligand(s) and because the protein, now present in a lipoparticle, can beused in assays where soluble proteins or whole cells cannot be used,such as assays where the protein of interest must be bound to a supportor substrate, including, but not limited to, an assay using amicrofluidic device, e.g., a biosensor assay.

[0094] However, the present invention is not limited to any particularassay. Rather, the present invention encompasses any assay where theprotein of interest is a membrane component and where study of thebinding of the protein with a ligand requires, or is facilitated by,presenting the protein in the context of a lipid bilayer and/orattaching the protein to a support or solid substrate. Such assaysinclude, but are not limited to, assays using a microfluidic device,e.g., an optical biosensor, PATIR-FTIR spectroscopy, which is a type ofbiosensor using total internal reflection Fourier-transform infraredspectroscopy (1998, Chem. Phys. Lipids 96:69-80), CPRW Biosensor(Coupled plasmon-waveguide resonance (CPWR) spectroscopy as described inSalamon et al. (1997, Biophys J. 73:2791-2197) and Salamon et al. (1998,Biophys J. 75:1874-1885), Multipole Coupling Spectroscopy (MCS) asdescribed in Signature Biosciences, www.signaturebio.com, Fiber opticbiosensors (Illumina) as described in Walt (2000, Science 287:451-452)and Dickinson et al. (1996, Nature 382:697-700), Michaels (1998,Analytical Chemisty 70:1242-1248), Lab-on-a-chip microfluidics(manufactured by, e.g., Caliper and Aclara) as described in Sundberg etal. (Current Opin. in Biotech. 11:47-53), and Bousse et al. (1999,Electrokinetic Microfluidic Systems, SPIE Microfluidic Devices andSystems II 3877:2-8, 9/20/99-9/21/99), Microchannels (Gyros'microchannels etched into a Compact Disc-based device) as described inwww.gyros.com, Microcantilevers (Protiveris) as described in Tamayo etal., 2001, Ultramicroscopy. 86:167-173), Wu et al. (2001, NatureBiotechnol. 19:856-860), Confocal microscopy and nanowell detection asdescribed in Hunt et al. (International Publication No. WO 01/02551),and Microwell binding assays. The aforementioned, as well as similarassays known in the art or to be developed in the future, areencompassed in the invention.

[0095] As more fully set forth elsewhere herein, the lipoparticle of theinvention, comprising a membrane protein of interest, can be used in awide variety of applications as would be appreciated by the skilledartisan once armed with the teachings disclosed herein. Moreparticularly, the lipoparticle can be used in assays relating to, forexample, but not limited to, drug screening, peptide screening, agonistversus antagonist discrimination, ADMET studies, structure-activityrelationships studies, vaccine development, food testing, chemicalsensing, light sensing, content release, monoclonal antibody production,fusion studies, phage display methods, ligand “fishing” oridentification, protein interaction mapping, various diagnostics, andproduction of artificial cells, among many others. Such uses would beunderstood by the skilled artisan to be encompassed in the inventionbased upon the disclosure provided herein.

[0096] The combination of the enveloped virus vector of the inventionwith a pharmaceutically acceptable carrier is specifically contemplatedas a method for providing the enveloped virus vector of the invention toa human.

[0097] The pharmaceutical compositions useful for practicing theinvention may be administered to deliver a dose of between 1 nanogramsper kilogram per day and 100 milligrams per kilogram per day of theenveloped virus vector.

[0098] Pharmaceutical compositions that are useful in the methods of theinvention may be administered systemically in oral solid formulations,ophthalmic, suppository, aerosol, topical, intravenous, or other similarformulations. In addition to the enveloped virus vector, suchpharmaceutical compositions may contain pharmaceutically acceptablecarriers and other ingredients known to enhance and facilitate envelopedvirus vector administration. Other possible formulations, such asnanoparticles and liposomes may also be used to administer an envelopedvirus vector according to the methods of the invention.

[0099] It is contemplated that the enveloped virus vector may be used todeliver an additional component to a cell for therapeutic, diagnostic,or prophylactic purposes. By way of example, the methods describedherein may be used to generate an enveloped virus vector wherein theluciferase gene present in NL-R⁻E⁻luc provirus used in Example 1 isreplaced, using methods well known in the art, with a gene encoding aprotein having cytotoxic or therapeutic properties. The resultingenveloped virus vector may be used to selectively kill or treat,respectively, HIV-infected cells in a patient to whom the resultingenveloped virus vector is administered.

[0100] As demonstrated by the specific embodiments described herein, oneskilled in the art may use the methods described herein to construct awide variety of enveloped virus vectors, each having an envelope whichcomprises at least one cellular virus receptor protein. By way ofexample, cells infected with RSV express EnvA, a viral envelope proteinof RSV, on their surface. An enveloped virus vector having an envelopewhich comprises Tva, the cellular virus receptor protein which iscognate to EnvA, may be used to infect cells which are infected withRSV. Similarly, an enveloped virus vector having an envelope whichcomprises CD4 and CCR5 may be used to infect cells which are infectedwith HIV.

[0101] In one embodiment, the enveloped virus vector is designed tospecifically target cells infected with a particular enveloped virus. Inthis embodiment, the cellular virus receptor protein of the envelopedvirus vector is cognate to a viral envelope protein of the particularenveloped virus. The particular enveloped virus may be any envelopedvirus, including, but not limited to, a retrovirus, a herpesvirus, arhabdovirus, human immunodeficiency virus (HIV), simian immunodeficiencyvirus (SIV), vesicular stomatitis virus, Rous sarcoma virus (RSV),murine leukemia virus (MLV), and the like. The cellular virus receptorprotein may be selected from the group consisting of CD4, CCR5, CXCR4,ICAM-1, ICAM-2, ICAM-3, CR3, CR4, CD43, CD44, CD46, CD55, CD59, CD63,CD71, a chemokine receptor, Tva, and MCAT-1. The cellular virus receptorprotein is preferably CD4, CCR5, CXCR4, Tva, or MCAT-1. As describedherein, the enveloped virus may further comprise an additional componentselected from the group consisting of a nucleic acid, an antisensenucleic acid, a gene, a protein, a peptide, Vpr protein, an enzyme, anintracellular antagonist of HIV, a radionucleotide, a cytotoxiccompound, an antiviral agent, and an imaging agent.

[0102] In some instances, it is desirable to have an enveloped virusvector of the invention which comprises a plurality of cellular virusreceptor proteins. For example, it may be desirable to target more thanone type of cell, each different type of cell being susceptible toinfection by a different enveloped virus. It may also be desirable tospecifically target cells which express more than a one viral envelopeprotein on the surface thereof, or wherein more than one cellular virusreceptor protein is necessary for attachment of an enveloped virus toand fusion of the envelope of the virus with the outer membrane of thecell. Or the proteins form a complex, quaternary structure (e.g. homo-or hetero-oligomers) that is useful for drug discovery targeting.

[0103] Thus, the enveloped virus vector of the invention includes, butis not limited to, an enveloped virus vector, as described herein,comprising a plurality of cellular virus receptor proteins. If a firstcellular virus receptor protein is cognate to a first viral envelopeprotein and the second cellular virus receptor protein is cognate to asecond viral envelope protein, then an enveloped virus vector comprisinga first cellular virus receptor protein and a second cellular virusreceptor protein may be used to target a cell which comprises the firstviral envelope protein, a cell which comprises the second viral envelopeprotein, or both of these cells. The vector may also be used to targetcells which comprise both the first viral envelope protein and thesecond viral envelope protein.

[0104] In one embodiment, each of the plurality of viral envelopeproteins is a retroviral envelope protein. In another embodiment, eachof the plurality of viral envelope proteins is a different viralenvelope protein which is expressed by a single retrovirus, preferablyHIV or SIV. In another embodiment, each of the plurality of cellularvirus receptor proteins is selected from the group consisting of CD4,CCR5, CXCR4, ICAM-1, ICAM-2, ICAM-3, CR3, CR4, CD43, CD44, CD46, CD55,CD59, CD63, CD71, a chemokine receptor, Tva, and MCAT-1. In one aspect,the enveloped virus vector comprises CD4 and a chemokine receptor. Inanother aspect, the enveloped virus vector comprises CD4 and CCR5. Inyet another aspect, the enveloped virus vector comprises CD4 and CXCR4.Other embodiments of the enveloped virus vector of the inventioninclude, but are not limited to, those in which the enveloped virusvector further comprises a replication-deficient HIV-1 virion,preferably an NL-R⁻E⁻luc virion, and those in which the enveloped virusvector comprises an additional component selected from the groupconsisting of a nucleic acid, an antisense nucleic acid, a gene, aprotein, a peptide, Vpr protein, an enzyme, an intracellular antagonistof HIV, a radionucleotide, a cytotoxic compound, an antiviral agent, andan imaging agent. In another aspect, the enveloped virusvector/lipoparticle of the invention, comprises a membrane cellularprotein of interest. Preferably, a lipoparticle comprises a multiplemembrane spanning protein that spans the lipid bilayer at least twice.The skilled artisan would understand, based upon the disclosure providedherein, that the invention encompasses a wide plethora of such complexproteins which traverse a membrane multiple times. To date, methods toincorporate such complex membrane spanning peptides in a simple bilayerhave not resulted in proteins where the function, structure, or both, ofthe peptide are maintained relative to the function and/or structure ofthe membrane protein in its natural state, e.g., in vivo. Further, theinvention provides a novel system comprising a lipid bilayer where oneor more such proteins can interact to form, e.g., homo and/orheterodimers, or otherwise interact with other membrane proteins oritself similarly to such interactions in the native membrane where theproteins typically reside.

[0105] The skilled artisan would appreciate, based upon the disclosureprovided herein, that multiple membrane spanning protein include, butare not limited to, G-protein couple receptors (GPCRs) (also known as7-transmembrane receptors, 7TM) that span the membrane seven times,e.g., CCR5, CXCR4, CCR3, mu-opioid receptor, as well as transporters(proteins that transport molecules such as, but not limited to, aminoacids or carbohydrates, across a membrane), ion channels, and the like.However, CD63, a multiple membrane spanning protein that has beenpreviously identified to be present as, without wishing to be bound byany particular theory, a contaminant associated with a virus membrane,is not encompassed in the invention.

[0106] In another aspect, the invention includes a compositioncomprising a lipoparticle comprising a single membrane spanning protein.Membrane proteins that span the membrane once include, but are notlimited to, CD4 (receptor for HIV and classic immunological modulator),neuropilin-1 (a VEGF receptor subtype), plexin-2 (involved in Rho/Racsignaling pathways), Tva (a receptor for Rous sarcoma virus, RSV, havinghomology to LDL-receptor), and the like. The lipoparticle is attached toa sensor surface, where a “sensor surface” is any substrate where achange in a property of the substrate mediated by the contacting of thesurface with a molecule or compound is detected and can be compared tothe surface in the absence of such contacting.

[0107] While the sensor surface can be a biosensor chip as exemplifiedherein, the sensor surface is not limited to such a chip. Instead, theskilled artisan would appreciate, based on the disclosure providedherein, that a sensor surface of the invention includes not only anybiosensor chip that is disclosed herein (e.g., a Biacore C1 chip, a F 1chip, and the like), known in the art, or to be developed in the future.Such sensor surfaces include, but are not limited to, a glass substratecomprising a coating of, e.g., gold, which can further comprise, forinstance, a dextran matrix. However, the invention is not limited to anyparticular sensor surface. The important feature of such a surface isthat a change in a characteristic of the sensor surface e.g., itsrefractive index, can be detected, preferably by an instrument connectedto the sensor surface, such that data or information from the sensor canbe assessed thus detecting the change, or lack of change, of thecharacteristic of the surface.

[0108] As noted elsewhere herein, the invention encompasses alipoparticle comprising a variety of membrane proteins, includingcombinations of proteins, which can form complexes when present in thelipoparticle. Such complexes include, but are not limited to, complexesof proteins that function together, e.g., CD4 and CCR5, CD4 and CXCR4,and the like. The lipoparticle can comprise MCAT-1, an amino acidtransporter that spans the membrane 14 times, and ion channels that spanthe membrane 6 times, e.g., K-channel KCNH2. All of these proteins havea similar feature: they span the membrane at least once. Many of thesespan the membrane multiple times. Proteins that span the membranemultiple times generally can not be removed from the lipid bilayerwithout destroying their structure. Anyone skilled in the art wouldunderstand that these proteins are difficult or impossible to isolateaway from a lipid membrane while still retaining their structuralintegrity.

[0109] In addition, some complexes (e.g. homo and hetero-oligomers)interact only when in the lipid membrane and so retain their quaternarystructure (formed by multiple subunits or proteins) only when in thelipid membrane. The lipoparticle of the invention encompassesincorporation of these proteins as well. In addition to these membraneproteins, which have been exemplified herein, other protein types andcategories are encompassed in the invention and include, but are notlimited to, the following: cytoplasmic domains of proteins (e.g., activeconformations, G-protein coupling domains, kinase motifs of proteinssuch as EGF-receptor, and the like); intracellular proteins associatedwith an intracellular membrane (e.g., nuclear transporters,mitochondrial receptors, endoplasmic reticulum and Golgi membraneproteins); and multimeric complexes (e.g., dimers and trimers, viralenvelope proteins, hetero-oligomers, and the like). More specifically,membrane proteins of the invention include, but are not limited to,GPCRs (e.g., CCR8, XCR1, CX3CR1), transporters (e.g., glucosetransporter), ion channels (e.g., K-channel Kv1.3 tetramers), tetramericType II protein (e.g., DC-SIGN tetramers), constitutively active GPCRs(e.g., HHV8 ORF74), viral proteins (e.g., HIV gp160, hepatitis C E1-E2Envelope protein, expressed on endoplasmic reticulum membrane).

[0110] Generation of the Enveloped Virus Vector of the Invention

[0111] In essence, the method of making the enveloped virusvector/lipoparticle of the invention involves formation of a virus-likeparticle using a cell which comprises a cellular virus receptor protein.Hence, the method of making the enveloped virus vector of the inventionrequires expression of at least a competent portion of the genome of anenveloped virus in a cell which comprises a cellular virus receptorprotein. The cellular virus receptor protein may be a normal componentof the cell or it may be provided exogenously to the cell using, forexample, known molecular biology techniques.

[0112] In one example of making the enveloped virus vector of theinvention, at least a competent portion of the genome of an envelopedvirus is provided to a producer cell which comprises a cellular virusreceptor protein, and the producer cell is thereafter incubated underconditions which permit expression of the gene products encoded by thecompetent portion of the genome. These gene products include factorswhich facilitate packaging of the competent portion of the genome into avirus capsid-like particle, association of the capsid-like particle withthe cell membrane, and budding of an enveloped virus-like particlecomprising the cellular virus receptor protein from the cell, wherebythe enveloped virus vector of the invention is thus generated.

[0113] In this example of making the enveloped virus vector of theinvention, the identity of the producer cell which is used is notcritical. However, the producer cell must comprise the cellular virusreceptor protein or the cellular membrane protein of interest, and thecompetent portion of the genome must enable formation of virus-likeparticles when expressed in the producer cell.

[0114] The manner of providing the competent portion of the genome ofthe enveloped virus to the producer cell is also not critical. However,when the competent portion of the genome is expressed in the cell, theformation of at least one enveloped virus-like particle must be enabled.The competent portion of the genome may be provided in the form of, forexample, the genome of an enveloped virus, a plasmid, or anon-circularized nucleic acid. The competent portion of the genome maybe, but is not limited to, a single-stranded RNA molecule, adouble-stranded RNA molecule, a single-stranded DNA molecule, adouble-stranded DNA molecule, or an RNA-DNA hybrid molecule. Theenveloped virus may be any enveloped virus, and is preferably aretrovirus. Preferred enveloped viruses are selected from the groupconsisting of HIV, SIV, RSV, and (ecotropic and amphotropic really referto the Envelope protein of MLV, not the core) MLV.

[0115] Conditions which enable formation of the enveloped virus vectorof the invention are well known in the art. These conditions may varydepending upon the properties of the producer cell and the envelopedvirus used. A number of references exist which describe conditions whichare useful for culturing particular enveloped viruses (Fields Virology,3rd ed., Fields et al., eds., Lippincott-Raven Publishers, Philadelphia,Pa.). Particular non-limiting examples are provided herein of conditionswhich are useful to enable formation of the enveloped virus vector ofthe invention.

[0116] Conditions which enable formation of the enveloped virus vectorof the invention include conditions which enable expression of thecompetent portion of the genome of the enveloped virus, conditions underwhich a cellular virus receptor protein is present in the membrane ofthe producer cell, and conditions which enable the formation ofenveloped virus-like particles from the components of a producer cellwhich has been provided with the competent portion of the genome.Further details regarding processes by which enveloped viral particlesare formed following provision to a cell of a competent portion of thegenome of an enveloped virus have been described in the art, forinstance by Wiley (1985, in Virology, Fields et al., ed., Raven Press,New York, 45-52).

[0117] Another example of making the enveloped virus vector of theinvention, further comprises providing an additional component to theproducer cell, whereby, upon formation of the enveloped virus vector,the enveloped virus vector comprises the additional component. Theadditional component may be any molecule which can be provided to thecytoplasm or the membrane of the producer cell. By way of example, theadditional component may be a nucleic acid, an antisense nucleic acid, agene, a protein, a peptide, Vpr protein, an enzyme, an intracellularantagonist of HIV, a radionuclide, a cytotoxic compound, an antiviralagent, an imaging agent, or the like.

[0118] Inclusion of the additional component in the enveloped virusvector of the invention may be accomplished by directly coupling theadditional component to the competent portion of the genome of theenveloped virus. For instance, if the competent portion of the genome isprovided to the producer cell in the form of a plasmid, the plasmid maycomprise a gene encoding an imaging agent, such as luciferase.

[0119] Inclusion of the additional component in the enveloped virusvector of the invention may also be accomplished by directly couplingthe additional component to a nucleic acid encoding the cellular virusreceptor protein. For example, if the cellular virus receptor protein isprovided to the producer cell in the form of a DNA molecule encoding thesame, an additional component comprising a protein may be provided tothe producer cell by including the sequence of a gene encoding theprotein in the DNA molecule, prior to provision thereof to the producercell.

[0120] The additional component may also be provided directly to themembrane or the cytoplasm of the producer cell by, for example,including the additional component in the extracellular medium of theproducer cell.

[0121] The producer cell need not normally comprise the desired cellularvirus receptor protein or membrane protein of interest. Thus, in anotherexample of making the enveloped virus vector of the invention, aproducer cell is provided with at least a competent portion of thegenome of an enveloped virus and a cellular virus receptorprotein/membrane protein of interest, and is thereafter incubated underconditions which permit formation of an enveloped virus vector of theinvention comprising the cellular virus receptor protein/membraneprotein. This method, therefore, does not employ a producer cell whichnaturally comprises the cellular virus receptor protein or membraneprotein of interest.

[0122] In this example of making the enveloped virus vector of theinvention, the manner of providing the cellular virus receptor proteinto the producer cell is not critical. By way of example, the cellularvirus receptor protein may be provided to the producer cell in the formof a protein associated with the membrane portion of a membrane vesicle,a protein associated with a liposome, a protein associated with themembrane of a cell, a membrane-free solution of the protein, a solidprotein, a protein associated with the envelope of an enveloped virus, aprotein associated with the envelope of an enveloped virus vector of theinvention, a nucleic acid, such as DNA or RNA, encoding the protein, avirus, which may be enveloped or non-enveloped, having a nucleic acidwhich encodes the protein, an enveloped virus vector having a nucleicacid which encodes the protein, or the like. Preferably, the cellularvirus receptor protein is provided to the producer cell in the form of aDNA molecule encoding the protein, more preferably in the form of aplasmid. Methods for delivering proteins, membrane vesicles, liposomes,nucleic acids, and viruses to a cell are described in the literature.These methods may be easily adapted to the present situation.

[0123] The identity of the cellular virus receptor protein is notcritical, except that it should be one which is cognate to a viralenvelope protein which is displayed on the surface of a cell with whichit is desired to fuse the enveloped virus vector, where applicable. Thecellular virus receptor protein may be any protein which is cognate to aviral envelope protein. Preferably, the cellular virus receptor proteinis cognate to a retroviral envelope protein, more preferably, it iscognate to a viral envelope protein of a virus selected from the groupconsisting of HIV, SIV, RSV, and ecotropic MLV. Also preferably, thecellular virus receptor protein is selected from the group consisting ofCD4, CCR5, CXCR4, ICAM-1, ICAM-2, ICAM-3, CR3, CR4, CD43, CD44, CD46,CD55, CD59, CD63, CD71, a chemokine receptor, Tva, and MCAT-1. Morepreferably, the first virus receptor protein is selected from the groupconsisting of CD4, CCR5, CXCR4, Tva, and MCAT-1.

[0124] A plurality of cellular virus receptor proteins may be providedto the producer cell in the same manner as that in which a singlecellular virus receptor protein is provided. When more than one cellularvirus receptor protein are provided to the producer cell, it ispreferred that one is CD4 and another is a chemokine receptor. Morepreferably, one is CD4 and another is CCR5 or CXCR4.

[0125] Further, in addition to the aforementioned proteins, theinvention encompasses embedding any protein of interest in thelipoparticles of the invention. Not only can the lipoparticles comprisea chemokine coreceptor, e.g., CCR5, CXCR4, CD4, neuropilin, or MCAT-1,or other membrane protein, but the invention includes lipoparticlescomprising any protein of interest, preferably a membrane component,that interacts with another protein. Such proteins include, but are notlimited to, any of the G-protein coupled receptors (GCPRs), atransporter, an ion channel, a type I membrane protein, a type IImembrane protein, and the like.

[0126] The skilled artisan would appreciate, based upon the disclosureprovided herein, that the lipoparticle comprises a multiple membranespanning protein which is not CD63. Moreover, the invention furtherincludes a composition comprising a lipoparticle comprising a proteinthat spans a membrane at least once, where the lipoparticle is attachedto a sensor surface. Such composition thus includes a protein spanningthe membrane at least and can comprise various proteins some spanningthe membrane once while other span the membrane at least twice. Suchproteins can interact to form complexes or otherwise interact whilepresent in the lipoparticle lipid bilayer.

[0127] In addition to a lipoparticle comprising a virus core, thepresent invention includes a lipoparticle comprising a bead core, alsoreferred to as a proteoliposome, where the lipoparticle is attached to asensor surface. A proteoliposome, as the term is used herein, comprisesa synthetic bead, e.g., plastic, surrounded by a membrane as describedby Mirzabekov et, al. Thus, the present invention is not limited tovirus-based lipoparticles, but includes such lipoparticles asproteoliposomes.

[0128] The lipoparticle can comprise non-membrane proteins. Moreparticularly, a lipoparticle can comprise water soluble proteins thatinteract with a membrane receptor of interest. For example, alipoparticle comprising a GCPR can be made with or without G-proteins,the intracellular subunits (alpha, beta, gamma) that couple to thereceptor and mediate signaling. These intracellular proteins caninfluence extracellular protein structure and can be important forformation of lipoparticles comprising complex membrane proteins thatinteract with soluble intracellular proteins.

[0129] Use of the Enveloped Virus Vector of the Invention

[0130] Targeted Vector

[0131] The enveloped virus vector of the invention may be used todeliver a composition to a target cell. This composition delivery methodis particularly useful when it is desired to deliver a compositionspecifically to a cell which comprises a viral envelope protein on itssurface. Specific examples of such a target cell include, but are notlimited to, a cell infected with an enveloped virus, such as HIV, SIV,RSV, or ecotropic MLV. A target cell may also be a cell infected withanother enveloped virus vector of the invention or a cell which hasfused with an enveloped virus vector other than the enveloped virusvector of the invention.

[0132] The composition to be delivered to the target cell by theenveloped virus vector of the invention may be any composition which maybe associated with the enveloped virus vector before, during, or aftergeneration of the enveloped virus vector. Examples of such compositionsinclude, but are not limited to, a nucleic acid, an antisense nucleicacid, a gene, a protein, a peptide, Vpr protein, an enzyme, anintracellular antagonist of HIV, a radionuclide, a cytotoxic compound,an antiviral agent, an imaging agent, and the like. The composition maybe associated with the enveloped virus vector by generating the vectorin a producer cell which comprises the composition, whereby, uponbudding of the enveloped virus vector of the invention from the producercell, the enveloped virus vector comprises the composition. Thecomposition may also be associated with the enveloped virus vector bytreating the vector with the composition after the vector has been made.Methods of associating a composition with an enveloped virus vectorinclude electroporation, specific adhesion of the composition to acomponent of the enveloped virus vector such as an envelope proteinthereof, and other methods known to one skilled in the art.

[0133] A particularly useful method of using the enveloped virus vectorof the invention relates to delivering a composition to a human cellinfected with HIV. Preferably, the HIV is HIV-1. Also preferably, thecomposition is a cytotoxic compound or an antiviral agent. The envelopedvirus vector which is useful in this method comprises CD4, a cellularvirus receptor protein for HIV-1, and either CCR5 or CXCR4, two othercellular virus receptor proteins for HIV-1. The composition may be, butis not limited to, an known anti-HIV agent, such as AZT, ddC, ddl, anHIV protease inhibitor, a cytotoxic agent, an enzyme, or a gene encodingan enzyme capable of activating small molecules in a cell, whichactivated small molecules are useful as cytotoxic or antiviral agents.Such enzymes include, but are not limited to, herpesvirus thymidinekinase, which is capable of phosphorylating gancyclovir, therebygenerating phosphorylated gancyclovir which is a cytotoxic agent. Hence,the enveloped virus vector comprising the composition, e.g. geneencoding an enzyme, CD4, and either of CCR5 or CXCR4 specificallydelivers the composition to human cells which are infected with HIV-1and may thereby kill the cell or prevent virus replication in that cell.Any number of other potential antiviral or cytotoxic agents which havebeen described in the literature may be so delivered using the envelopedvirus vector of the invention.

[0134] The enveloped virus vector useful for delivering a composition toa target cell may comprise a single cellular virus receptor protein or aplurality of cellular virus receptor proteins. By way of example, theenveloped virus vector may comprise a cellular virus receptor proteinselected from the group consisting of CD4, CCR5, CXCR4, ICAM-1, ICAM-2,ICAM-3, CR3, CR4, CD43, CD44, CD46, CD55, CD59, CD63, CD71, a chemokinereceptor, Tva, and MCAT-1. In preferred embodiments, the enveloped virusvector comprises Tva, MCAT-1, CD4, CCR5, CXCR4, both CD4 and CCR5, orboth CD4 and CXCR4. However, the invention is not limited to thesemolecules. Indeed, the data disclosed herein demonstrating thesuccessful incorporation of MCAT-1 into MLV (murine leukemia virus)lipoparticles, demonstrate that type 1 (i.e., single-spanning proteins),and not just multiple membrane spanning proteins such as GCPRs, can beembedded in the lipoparticles while preserving their native bindingability. Preservation of binding ability relative to the protein astypically present in the cell membrane can be assessed by functionalassays, such as, but not limited to, the fusion assays disclosed herein,and also by using biosensor assays such as those exemplified herein.

[0135] The enveloped virus vector of the invention may be used to expandthe host range or tissue tropism of an enveloped virus or vector. Thehost range and tissue tropism of an enveloped virus or vector isdetermined in part by the protein composition of the envelopesurrounding the virus or vector. Thus, the enveloped virus vector may beused to provide a protein or another component to a target cell prior toproduction of another enveloped virus or vector using the same targetcell. By way of example, a first enveloped virus vector may be used toprovide a cellular protein to a cell. When a second enveloped virusvector or enveloped virus subsequently enters the cell, the cellularprotein becomes a component of the second enveloped virus vector or theenveloped virus upon replication of the same within the cell. Thecellular protein may be one which alters the tissue tropism of thesecond vector or virus thereby expanding the host range of that secondvector or virus.

[0136] To alter the host range or tissue tropism of an enveloped virusor vector, a target cell is contacted with the enveloped virus vector ofthe invention, the envelope of which comprises a tropism determinant,preferably a cellular virus receptor protein or a viral envelopeprotein, wherein the enveloped virus or vector is capable of fusing withthe target cell. Following fusion of the enveloped virus vector and thetarget cell, the membrane of the target cell comprises the tropismdeterminant provided by the enveloped virus vector of the invention.Subsequent use of the target cell to produce the enveloped virus orvector results in the inclusion of the tropism determinant in theenvelope of the enveloped virus or vector, thereby altering the tissuetropism of the enveloped virus or vector.

[0137] An aspect of the invention is illustrated in FIG. 7. The diagramin FIG. 7, Panel A, depicts normal infection of a cell (ellipseenclosing a filled ellipse) by enveloped viruses having envelopes(circles enclosing shaded hexagons) which comprise viral envelopeproteins (lines extending from viral envelopes and each having a filledcircle at the distal end thereof). In the upper image, virions assemble,bud from the cell, and incorporate viral envelope proteins into theenvelope of the virion. In the center image, virions infect target cellsthat express a cellular virus receptor protein (lines extending fromcell and each having a hemicircle at the distal end thereof). In thelower image, virions assemble, bud from the cell, and incorporate viralenvelope glycoproteins into the envelope of the virion.

[0138] The diagram in FIG. 7, Panel B, depicts a method of altering thetropism of an enveloped virus. In the upper image, enveloped virusvectors (circles enclosing unshaded hexagons) assemble, bud from thefirst cell (irregular shaped body enclosing a filled ellipse), andincorporate into their envelopes both a first viral envelope protein(lines extending from viral envelopes and each having an open circle atthe distal end thereof) and cellular virus receptor proteins (linesextending from the first cell and each having a hemicircle at the distalend thereof) cognate to a second viral envelope protein. In the centerimage, the enveloped virus vectors are capable of infecting a secondcell (ellipse enclosing a filled ellipse) which is itself infected by anenveloped virus (circles enclosing shaded hexagons). The second cellexpress the second viral envelope protein (lines extending from thesecond cell and each having a filled circle at the distal end thereof)and the cellular virus receptor protein is capable of binding to thesesecond viral envelope proteins. In the lower image, following infectionof the second cell by enveloped virus vectors, altered enveloped virusesare assembled, bud from the second cell, and incorporate into theirenvelopes both the first viral envelope protein and the second viralenvelope protein. Thus, the tropism of the altered enveloped virusdiffers from that of the original enveloped virus.

[0139] By way of example, the host range of an enveloped murinecytotoxic virus which is normally not capable of infecting any type ofhuman cells may be altered such that the virus is capable of infectingHIV-infected human cells. To alter the cytotoxic virus, a target cellsuch as a murine T-cell which the cytotoxic virus is normally capable ofinfecting, is contacted with the enveloped virus vector of theinvention. This enveloped virus vector comprises the cellular virusreceptor proteins CD4 and CXCR4, and a viral envelope protein to which acellular virus receptor protein of the target cell is cognate, whereinthe enveloped virus vector is capable of fusing with the target cell.Following fusion, the target cell membrane comprises CD4 and CXCR4.Subsequent infection of the target cell by the murine virus andreplication of the virus therein results in the production of apseudotyped cytotoxic virus particle having CD4 and CXCR4. Infection ofa human infected with HIV-1 with a pseudotyped cytotoxic viruscomprising CD4 and CXCR4 results in death of HIV-1-infected human cells.Human cells which are not infected with HIV-1 are not killed, becausethey cannot be infected with the altered cytotoxic virus.

[0140] Other properties of an enveloped virus or virus vector having anenvelope may be altered by including one or more additional componentsin the enveloped virus vector of the invention. Fusion of the envelopedvirus vector with a target cell provides the additional component to thetarget cell. Subsequent use of the target cell to produce an envelopedvirus or vector results in the inclusion of the additional component inthe virus or vector. The selection of the additional component isdependent upon the alteration which is desired. Numerous components maybe selected which are known in the art to affect a property of a virusin a desired manner. By way of example, the additional component may beselected from the group consisting of a nucleic acid, an antisensenucleic acid, a gene, a protein, a peptide, Vpr protein, an enzyme, anintracellular antagonist of HIV, a radionuclide, a cytotoxic compound,an antiviral agent, an imaging agent, and the like.

[0141] The enveloped virus vector of the invention may be used to rendera cell susceptible to fusion with an enveloped virus or vector, whereinthe cell is not normally susceptible to such fusion. Thus, it ispossible to generate a non-human animal model of a viral disease ofhumans and to enable staged delivery of enveloped viral vectors to ahuman cell.

[0142] A non-human animal model of a human disease may be made bycontacting a cell of the non-human animal with an enveloped virus vectorcomprising a cellular virus receptor protein which is cognate to a viralenvelope protein of a human pathogenic enveloped virus, wherein theenveloped virus vector is capable of fusing with the cell of thenon-human animal. Fusion of the vector and the cell provides the cellwith the cellular virus receptor protein, whereby the cell becomessusceptible to infection by the human pathogenic enveloped virus. Thenon-human animal cell which becomes susceptible to infection by thehuman pathogenic enveloped virus may be used both in vitro or in vivo toinvestigate the disease mediated by the pathogenic virus withoutexposing a human subject to the virus.

[0143] By way of example, a murine model of HIV-1 infection may begenerated by contacting a murine T-cell with an enveloped virus vectorwhich comprises a viral envelope protein to which a murine cellularvirus receptor protein normally found on the surface of the murineT-cell is cognate and a cellular virus receptor protein which is cognateto a viral envelope protein of HIV-1. By virtue of the interactionbetween the murine cellular virus receptor protein and the viralenvelope protein of the vector, the vector is capable of fusing with thecell and thereby provides to the cell the cellular virus receptorprotein which is cognate to a viral envelope protein of HIV-1. As aresult, the murine T-cell is rendered susceptible to infection by HIV-1.

[0144] In a preferred method, the enveloped virus vector comprises aplurality of cellular virus receptor proteins, each of which is cognateto a viral envelope protein of HIV. For example, an enveloped virusvector comprising CD4 and a chemokine receptor such as, but not limitedto, CCR5 and CXCR4, may be used.

[0145] Alternately, an analogous method may be used to render anon-human cell susceptible to infection by a non-human virus which issimilar to a human pathogenic virus. The alternate method substitutesthe non-human virus in place of the human pathogenic enveloped virusreferred to herein. By way of example, a cell of a non-human animal maybe rendered susceptible to infection by SIV, which is similar to HIV,using the methods described herein.

[0146] The enveloped virus vector of the invention may comprise both acellular virus receptor protein and a viral envelope protein. Hence, thevector may be used to enable staged delivery of enveloped viral vectorsto a cell. By way of example of such staged delivery, a single cell in apopulation of cells may be fused in a first stage with a first envelopedvirus vector which comprises a first viral envelope protein and a firstcellular virus receptor protein. Later, in a second stage, a secondenveloped virus vector may be provided to the population of cells, whichsecond vector comprises a second cellular virus receptor protein whichis cognate to the first viral envelope protein, whereby the single cellfuses with the second enveloped virus vector, while the remainder of thecells in the population do not. Likewise, the second enveloped virusvector may further comprise a second viral envelope protein, and a thirdenveloped virus vector comprising a third cellular virus receptorprotein which is cognate to the second viral envelope protein may befused with the single cell in a third stage. There is no theoreticallimit to the number of such stages which may be performed using themethods described herein.

[0147] Staged delivery of enveloped virus vectors to a cell has numeroususes including, but not limited to, staged delivery of therapeuticagents wherein agents having relatively low toxicity are delivered inearly stages while agents having greater toxicity are delivered in laterstages to cells which were not suitably treated in the early stages. Inaddition, delivery of a gene to a cell may be accomplished whereinexpression of the gene in the cell is desired for only a limited periodof time. The gene may be delivered to the cell in a first stage using anenveloped virus vector of the invention, and an agent which halts geneexpression or which kills the cell to which the gene was delivered inthe first stage may be delivered in a later stage using a secondenveloped virus of the invention.

[0148] The enveloped virus vector of the invention may also be used toproduce a cellular virus receptor protein to obtain quantities of theprotein which can be easily purified, relative to purification of theprotein from the physiological cellular source of the protein. Toaccomplish this, the enveloped virus vector of the invention isgenerated using any of the methods described herein. By separating theenveloped virus vector, which includes the cellular virus receptorprotein, from the cells used to make or propagate the vector, thecellular virus receptor protein is obtained. Furthermore, because viruspropagation may be performed on a large scale, propagation of theenveloped virus vector of the invention may be used to produce a largequantity of the cellular virus receptor protein. The vector maysubsequently be separated from the producer cell, whereby a largequantity of the cellular virus receptor protein may be biochemicallypurified from the enveloped virus vector using ordinary biochemical andimmunological technology to yield pure or substantially pure protein.

[0149] Substantially pure protein obtained as described herein may bepurified by following known procedures for protein purification, whereinan immunological, enzymatic or other assay is used to monitorpurification at each stage in the procedure. Protein purificationmethods are well known in the art, and are described, for example inDeutscher et al. (ed., 1990, Guide to Protein Purification, HarcourtBrace Jovanovich, San Diego).

[0150] As used herein, the term “substantially pure” describes acompound, e.g., a protein or polypeptide which has been separated fromcomponents which naturally accompany it. Typically, a compound issubstantially pure when at least 10%, more preferably at least 20%, morepreferably at least 50%, more preferably at least 60%, more preferablyat least 75%, more preferably at least 90%, and most preferably at least99% of the total material (by volume, by wet or dry weight, or by molepercent or mole fraction) in a sample is the compound of interest.Purity can be measured by any appropriate method, e.g., in the case ofpolypeptides by column chromatography, gel electrophoresis or HPLCanalysis. A compound, e.g., a protein, is also substantially purifiedwhen it is essentially free of naturally associated components or whenit is separated from the native contaminants which accompany it in itsnatural state.

[0151] Study of Protein Interaction

[0152] The enveloped virus vector of the invention may also be used toascertain whether a test protein is a viral envelope protein to which aparticular cellular virus receptor protein is cognate. To accomplishthis, a test cell is provided, comprising a control cell having the testprotein in the outer membrane thereof. An enveloped virus vector is alsoprovided, wherein the vector comprises the desired cellular virusreceptor protein. The enveloped virus vector is contacted with the testcell, and the ability of the enveloped virus vector to fuse with thetest cell is assessed. The ability of the enveloped virus vector to fusewith the test cell is an indication that the test protein is a viralenvelope protein to which the certain cellular virus receptor protein iscognate.

[0153] The test protein may be provided to the test cell by infectingthe test cell with an enveloped virus which comprises the test protein,by providing to the test cell a nucleic acid which encodes the testprotein and which is capable of expression in the test cell, or by anyother method known to one of skill in the art of molecular biology.

[0154] The test protein may be any protein. Preferably, the test proteinis a membrane protein, more preferably a membrane protein present in themembrane of a cell infected with an enveloped virus. The test proteinmay also be a protein naturally present in the envelope of an envelopedvirus.

[0155] The ability of the enveloped virus vector to fuse with the testcell may be assessed in any manner known to one of skill in the art. Forexample, immunological methods are well known in the art of detectingviral infection of a cell. Preferably, the enveloped virus vectorfurther comprises an additional component, the presence of whichcomponent in the test cell may be easily determined. Non-limitingexamples of such additional components include luciferase protein, agene encoding luciferase protein, a radionuclide, and an imaging agent.

[0156] Indeed, protein-protein interaction can be assessed using methodsof such interaction that do not require cell fusion as an indication ofprotein-protein interaction. As exemplified herein, such methodsinclude, but are not limited to, contacting a virus lipoparticlecomprising a test protein with a second protein, e.g., a potentialligand or a known ligand the interaction of which is being assessed orinhibited, and assessing the interaction between the two proteins. Theskilled artisan would appreciate, based upon the disclosure providedherein, that the ligand, or potential ligand, can be any protein,membrane or otherwise, and that the ligand is also not limited to anyparticular protein. Thus, the ligand, or potential ligand, thelipoparticle is contacted with, i.e., the ligand, can include, amongother proteins, the same protein that is present on the lipoparticle, anantibody, a peptide, or a chemical compound that is not comprised ofamino acids, such as, but not limited to, a carbohydrate, a lectin, achemical low molecular weight substance. Such ligand encompasses thosedescribed in Doranz et al., 1997, J. Exp. Med. 186:1395-1400.) The term“ligand,” as used herein, encompasses any protein or compound that canbind with a protein present in a lipoparticle. The ligand encompasses aprotein or non-protein compound that can bind with a protein present ina lipoparticle.

[0157] “Lipoparticle,” as used herein, means a small particle of aboutone nanometer to about a micrometer in size, comprising an externallipid bilayer further comprising a protein. The core, or interior, ofthe lipoparticle is not a crucial feature of the invention.Lipoparticles of the invention include, but are not limited to, a virus,e.g., a retrovirus (e.g., HIV, MLV, RSV, VSV, and the like), a vesicularstomatitis virus, and the like), a membrane-enveloped virus, and aproteoliposome (i.e., a lipid bilayer formed around a bead where thebilayer comprises a protein of interest). The protein-proteininteraction can be assessed using any method either known in the art orto be developed for assessing protein-protein interaction. Such methodsinclude, but are not limited to, using a microfluidic device.Preferably, the microfluidic device comprises a microchannel and/or amicrowell.

[0158] Microfluidics is the miniaturization of biological separation andassay techniques to such a degree that multiple experiments can beaccomplished on a miniature scale. Tiny quantities of solvent, sample,and reagents, typically in the micrometer or nanoliter range, aresteered through narrow (typically micron scale) channels on the chip orplaced within miniature wells, where they are mixed and analyzed by suchtechniques as electrophoresis, fluorescence detection, immunoassay, orindeed almost any classical laboratory method (Nature Biotechnology,“Microfluidics—downsizing large-scale biology,” August 2001).

[0159] More preferably, the microfluidic device is a biosensor device.[Biosensor devices are designed to measure the interaction betweenbiological molecules. Typically, biosensors measure direct interactionsbetween a protein of interest and potential ligands (proteins,antibodies, peptides, small molecules) that may bind to it. Biosensorsare typically highly sensitive and can work with and detect even veryweak or very small quantity interactions. Biosensor devices have beenconstructed that consist of optical chips, fiber optics, spectrometerdetectors, microchannel chips, nanowells, and microcantilevers. Evenmore preferably, the assay comprises using a biosensor device whereinthe device is a surface plasmon resonance biosensor device.

[0160] The most commonly used optical biosensors (Biacore™) are based onsurface plasmon resonance (SPR) that measures changes in refractiveindex at the sensor surface (see, e.g., Canziani et al., 1999, Methods.19:253-269; Rich and Myszka, 2000, Curr. Opin. Biotechnol. 11:54-61.)Biacore's biosensor system measures interactions between the immobilizedmolecules on the surface of sensor chips and the molecules contained ina solution that passes over the surface under controlled flowconditions. The Biacore biosensor chip comprises a glass surface, coatedwith a thin layer of gold that provides the physical conditions requiredfor SPR, which is the basis by which changes in mass, and thereforebinding, on the sensor chip surface is detected. Gold-dextran sensorchips are further comprised of a dextran layer which covers the gold andis specifically designed to minimize non-specific binding and provide afavorable environment for interactions between biomolecules. SPR chipsuse microfluidic channels to deliver the sample on the chip in precisequantities and at precise times.

[0161] Optical detection technology with SPR measures changes in mass onthe sensor chip surface (i.e. binding), with a sensitivity of less thana picogram. SPR biosensors are unique in their ability to quantifybinding in real-time, thereby producing the following four data sets: 1)specificity of binding, 2) concentration, 3) kinetics (association anddissociation rates), and 4) affinity. Briefly, as exemplified elsewhereherein, the lipoparticle comprising a test protein is affixed onto asurface or solid support or substrate. The second protein is allowed tocontact the lipoparticle and the interaction, if any, between the testprotein and the second protein, which may be the same protein wherehomologous protein interaction is being assessed, is characterized usingan instrument.

[0162] One skilled in the art would appreciate, based upon thedisclosure provided herein, that the nature of the instrument, or theparticular surface to which the lipoparticle is attached, are notcrucial. That is, while a derivatized gold surface or a short carboxydetran matrix can be used to attach the lipoparticle thereto, theinvention is in no way limited to these surfaces; instead, the inventionincludes any surface that can be used in a microfluidic device to assessthe interaction of proteins. Such substrates include, but are notlimited to, a wide plethora of biosensor “chips” that are commerciallyavailable, and others surfaces that are known in the art, or suchsurfaces as will be developed in the future.

[0163] Biosensor surfaces for other biosensor and microfluidic devicesare composed of quartz glass (e.g. Caliper's LabChips), Acrylic(Aclara's mirofluidic chips), plastic (Gyros' compact discmicrofluidics), plastic wells (Evotec's nanowells and SignatureBioscience's spectroscopy platform are relatively surface independent sostandard 96-well plates made of polystyrene or polypropylene can beused), silver (CPWR biosensors use a silver instead of a gold surface),silicon (Protiveris' microcantilevers are composed of silicon), or glassfiber (Illumina's optical fiber biosensors). Each of these surfaces can,in turn, be modified for better coupling, lower background, increasedsignal, etc. Modifications of the biochip surface include attachment ofdextran, PEGylation, coating with BSA, etc.

[0164] One instrument useful for assessing molecule interactions, e.g.,an optical biosensor, can be used to detect any change in the refractiveindex at a sensor surface under various conditions. This allows thedetection of interactions between molecules as detected by changes inthe refractive index at the sensor surface. The change in the surfaceresonance indicates the interaction between binding partners, at leastone of which is tethered, or otherwise attached, to the surface of thesensor. More particularly, one molecule is tethered, the other is flowedover through microchannel. If the flowed molecule attaches to thetethered molecule, a signal is detected.

[0165] Methods of attaching a lipoparticle to a sensor surface are wellknown in the art and are exemplified elsewhere herein. Moreover, theinvention encompasses methods of attaching, including tethering, alipoparticle to a surface for measuring interaction of a protein presentin the lipoparticle with another protein. Such methods include, but arenot limited to, activation of amino acid carboxyl groups, and the like.Other attachment technologies are also encompassed in the inventionincluding a Biacore a class of chips, all designed to capture theprotein of interest (in this instance, the entire lipoparticle)differently. One chip, the LI chip, acts differently from the othercommercially available chips in that it is hydrophobic so is designed tocapture lipid vesicles by attracting the lipid to the chip surface andthen “melting” lipid vesicles onto the chip surface so that the endresult is that the LI chip surface is covered by a bilayer of lipid withthe protein of interest embedded in the bilayer. Such chips, and othersknown in the art or to be developed in the future, are included in thepresent invention.

[0166] The conditions used in the assay to asses protein interactionwith a ligand are not a limiting factor. That is, a wide plethora ofconditions for use in assays of binding interactions are known in theart and are not repeated herein. Further, the invention encompasses anyassay conditions developed in the future. Preferably, the pH forattachment of the lipoparticle to a sensor surface is about 5.5. Theskilled artisan would appreciate, based upon the disclosure providedherein, that the pH for attachment depends on the method of attachmentused, and can be from about pH 4 to 10. The number of RU captured isproportional to the number of RU signal that is obtained, which is, inessence, the sensitivity and signal:noise specificity of detection ofthe assay. Typically, the procedures exemplified herein capturedthousands (e.g. 2000-6000) of RU lipoparticle and obtained a signal ofseveral hundred RU.

[0167] Preferably, the bound lipoparticle can withstand at least oneregeneration cycle where bound ligands/analytes are removed withoutdamaging the lipoparticles or the protein that they comprise.Regeneration conditions are well known in the art, or are disclosedherein. However, the invention is in no way limited to these or anyother regeneration parameters, but encompasses any regeneration regimenknown in the art or to be developed in the future, including, but notlimited to, a brief pulse using a regeneration mixture comprising aboutequal parts of a pH 5 and a chaotropic solution.

[0168] The skilled artisan would understand, based upon the disclosureprovided herein, that the invention can encompass a lipoparticle formedaround a bead, e.g., a proteoliposome. Such lipoparticle/bead constructsare well known in the art and methods for the production of suchconstructs is set forth in, for instance, Mirzabekov et al. (2000,Nature Biotechnology 18: 649-654), and Babcock et al. (2001, J. Biol.Chem.) Briefly, proteoliposomes consist of nano- or micrometer sizedbeads that are surrounded by a lipid membrane bilayer that is embeddedwith membrane-bound receptors. To form proteoliposomes, the surface ofnonporous magnetic beads is covalently conjugated with an antibody thatrecognizes a C-terminal epitope tag (e.g. FLAG) on the receptor ofinterest. The beads are used to capture detergent-solubilized receptor(expressed at high levels in a cell line), washed, and then mixed withdetergent-solubilized lipid. During the removal of detergent bydialysis, the lipid bilayer membrane self-assembles around the beads andthe receptor is returned to its native environment. Proteoliposomes areuniform in size, stable in a broad range of harsh conditions (high orlow pH, extremes of ionic strength, ranges of temperature, 0-50° C.),and can be used in FACS and competition assays typically applied tocells. By using magnetic beads, proteoliposomes can be easily isolatedand purified.

[0169] It was previously demonstrated that complex membrane-boundreceptors (the GPCRs CCR5 and CXCR4) can be incorporated intoproteoliposomes (e.g., Nature Biotechnology (2000) and J. Biol. Chem.(2001)). These proteoliposomes were used to select CCR5-specificantibodies from a recombinant phage display library and for binding toantibodies and proteins. The skilled artisan would appreciate, basedupon the disclosure provided herein, that such proteoliposomes,comprising a membrane protein, can be attached to a sensor surface.

[0170] In a variation of this method, the test cell and a control cellare each contacted with the enveloped virus vector. The ability of theenveloped virus vector to fuse with the test cell is assessed, and theability of the enveloped virus vector to fuse with the control cell isassessed. A greater ability of the enveloped virus vector to fuse withthe test cell compared with the ability of the enveloped virus vector tofuse with the second control cell is an indication that the test proteinis a viral envelope protein to which the particular cellular virusreceptor protein is cognate.

[0171] In another variation of this method, a library comprising aplurality of nucleic acid-containing vectors is provided, each of whichcomprises a nucleic acid which corresponds to at least a portion of aviral nucleic acid, wherein when a vector selected from the library isprovided to a control cell, any protein encoded by the nucleic acid ofthat vector is capable of expression in the control cell. Methods ofmaking such vectors are well known to one skilled in the art ofmolecular biology and are described in such references as Sambrook etal. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York). An individual vector is provided to a controlcell and is expressed therein to generate a test cell. An envelopedvirus vector comprising the particular cellular virus receptor proteinis contacted with the test cell. If the nucleic acid encodes a viralenvelope protein to which the particular cellular virus receptor proteinis cognate, then the test cell is susceptible to fusion with theenveloped virus vector. The ability of the enveloped virus vector tofuse with the test cell thus indicates that the nucleic acid of thevector encodes a viral envelope protein to which the particular cellularvirus receptor protein is cognate. The nucleic acid of the vector maysubsequently be isolated, cloned, and characterized using techniqueswell known in the art.

[0172] The enveloped virus vector of the invention may further be usedto ascertain whether a test protein is a cellular virus receptor proteinwhich is cognate to a particular viral envelope protein. A test cell isprovided, comprising a control cell having the particular viral envelopeprotein in the outer membrane. An enveloped virus vector is alsoprovided, wherein the vector comprises the test protein in the envelope.The enveloped virus vector is contacted with the test cell, and theability of the enveloped virus vector to fuse with the test cell isassessed. The ability of the enveloped virus vector to fuse with thetest cell is an indication that the test protein is a cellular virusreceptor protein which is cognate to the particular viral envelopeprotein.

[0173] The viral envelope protein may be provided to the test cell byinfecting the test cell with an enveloped virus which comprises theparticular viral envelope protein, by providing to the test cell anucleic acid which encodes the particular viral envelope protein andwhich is capable of expression in the test cell, or by any other methodknown to one of skill in the art of molecular biology.

[0174] The enveloped virus vector may be made by any of the methodsdescribed herein for making an enveloped virus vector of the invention,by substituting the test protein in place of the cellular virus receptorprotein or by substituting a nucleic acid encoding the test protein inplace of the nucleic acid encoding the cellular virus receptor protein,as appropriate.

[0175] The test protein may be any protein. Preferably, the test proteinis a membrane protein, more preferably a membrane protein present in themembrane of a cell susceptible to infection by an enveloped virus. Thetest protein may also be a protein present in the envelope of anenveloped virus or in a virus vector having a membrane envelope. Morepreferably a membrane protein that spans the membrane multiple times.Most preferably any receptor found on the surface of a cell or on thesurface of intracellular organelles or intracellular membranes.

[0176] The ability of the enveloped virus vector to fuse with the testcell may be assessed in any manner known to one of skill in the art, asdescribed herein.

[0177] In a variation of this method, the test cell and a control cellare each contacted with the enveloped virus vector. The ability of theenveloped virus vector to fuse with the test cell is assessed. Theability of the enveloped virus vector to fuse with the control cell isassessed. A greater ability of the enveloped virus vector to fuse withthe test cell, compared with the ability of the enveloped virus vectorto fuse with the second control cell, is an indication that the testprotein is a cellular virus receptor protein which is cognate to theparticular viral envelope protein.

[0178] In another variation of this method, a library comprising aplurality of recombinant enveloped virus vectors is provided, each ofwhich vectors comprises a nucleic acid which corresponds to at least aportion of a cellular nucleic acid. When a producer cell is fused with arecombinant enveloped virus vector of the library, the nucleic acid iscapable of expression in the producer cell. Methods of making suchrecombinant enveloped virus vectors are well known to one skilled in theart of molecular biology and are described in such references asSambrook et al. (1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York). An individual vector is fused witha producer cell in which the vector is capable of replication and theportion of the cellular nucleic acid is expressed, yielding a progenyvector. Thus, if the nucleic acid encodes a cellular virus receptorprotein, the progeny vector comprises the cellular virus receptorprotein in its envelope. An individual progeny vector is contacted withthe test cell comprising the particular viral envelope protein, and theability of the progeny vector to fuse with the test cell is assessed.The ability of the progeny vector to fuse with the test cell indicatesthat the cellular nucleic acid encodes a cellular virus receptor proteinwhich is cognate to the particular viral envelope protein. The cellularnucleic acid which the progeny vector comprises may be isolated, cloned,and characterized using methods well known in the art.

[0179] By way of example, a test cell comprising EnvA, the viralenvelope protein of RSV(A), may be used to identify whether a testprotein is a cellular virus receptor protein which is cognate to EnvA.To accomplish this, a cellular genome obtained from a cell which issusceptible to infection by RSV(A) is cleaved to form cellular nucleicacids. Individual cellular nucleic acids are inserted into an envelopedvirus vector of the invention which is capable of fusing with andreplicating within a producer cell which is not susceptible to infectionby RSV(A). The vector comprising the cellular nucleic acid is fused witha producer cell and replicated therein, yielding a progeny vector. Thecellular nucleic acid, delivered to the producer cell via fusion withthe vector, is expressed by the producer cell, whereby any membraneprotein encoded by the cellular nucleic acid will be present in theenvelope of the progeny vector. The progeny vector is contacted with thetest cell, and the ability of the progeny vector to fuse with the testcell is an indication that the cellular nucleic acid encodes a cellularvirus receptor protein which is cognate to EnvA.

[0180] The enveloped virus vector of the invention may be used toinvestigate the ability of a composition to affect the interactionbetween a viral envelope protein and a cellular virus receptor proteinwhich is cognate to the viral envelope protein. To accomplish this, acell is provided which comprises the viral envelope protein. Anenveloped virus vector comprising a cellular virus receptor proteinwhich is cognate to the viral envelope protein is contacted with thecell in the presence or absence of the composition, and the ability ofthe enveloped virus vector to fuse with the cell is assessed. A greateror lesser ability of the enveloped virus vector to fuse with the cell inthe presence of the composition, relative to the ability of theenveloped virus vector to fuse with the cell in the absence of thecomposition, is an indication that the composition is capable ofaffecting the ability of the viral envelope protein and the cellularvirus receptor protein to interact.

[0181] The cell may be prepared by infecting cells with a viruscomprising the viral envelope protein, by providing the cell with anucleic acid which encodes the viral envelope protein and which iscapable of being expressed in the cell, or by any other method known toone of skill in the art of membrane protein biochemistry.

[0182] The enveloped virus vector may be prepared using any of themethods described herein. Preferably, the enveloped virus vector furthercomprises an additional component, the presence of which component inthe cell may be easily determined. Non-limiting examples of suchadditional components include luciferase protein, a gene encodingluciferase protein, a radionuclide, and an imaging agent.

[0183] One skilled in the art would appreciate, based on the disclosureprovided herein, that the lipoparticle can comprise any membraneprotein, i.e., any protein that typically is associated with a membrane.Preferably, the lipoparticle comprises a multiple membrane spanningprotein. That is, the protein spans the membrane at least twice. Suchmultiple membrane spanning proteins encompass a wide plethora ofmembrane proteins including, but not limited to, the 7 transmembranereceptor proteins (e.g., G-protein coupled receptor proteins, GPCRs,which include chemokine coreceptors), ion channels, transporters (suchas amino acid transporter MCAT-1 and glucose transporter, and the like).

[0184] Further, one skilled in the art would understand, based upon thedisclosure provided herein, that the invention includes a compositioncomprising a lipoparticle attached to a sensor surface further whereinthe liposome comprises a membrane spanning protein. The membranespanning protein encompasses any protein that spans the membrane atleast once. The skilled artisan would appreciate, based upon thedisclosure provided herein, that the lipoparticle of the invention canencompass a multiple membrane spanning protein that spans the membraneat least twice and is not CD63, and, when the lipoparticle is attachedto a sensor surface, it can further comprise a membrane spanning proteinthat spans at least once.

[0185] Kits

[0186] The invention includes various kits which comprise a lipoparticlecomprising a multiple membrane spanning protein, and/or compositions ofthe invention, an applicator, and instructional materials which describeuse of the compound to perform the methods of the invention. Althoughexemplary kits are described below, the contents of other useful kitswill be apparent to the skilled artisan in light of the presentdisclosure. Each of these kits is included within the invention.

[0187] In one aspect, the invention includes a kit for assessing thebinding interaction of a membrane spanning protein with a ligand. Thekit comprises a lipoparticle comprising membrane spanning protein, aligand of the membrane protein, and a substrate to which thelipoparticle can be attached. The kit further comprises an applicator,which applicator can be used to attach the lipoparticle to the substrateand/or for applying the ligand such that the ligand is contacted withthe lipoparticle comprising the membrane protein. Such an applicatorincludes, but is not limited to, a pipette, an injection device, adropper, and the like.

[0188] One skilled in the art will understand that the kit includes alipoparticle already attached to a substrate with or without the ligandbeing bound to the membrane protein. The substrate can then be examinedusing methods well known in the art to detect any change in thesubstrate mediated by or associated with the ligand binding with itscognate membrane receptor present in the lipoparticle.

[0189] Moreover, the kit comprises an applicator and an instructionalmaterial for the use of the kit. These instructions simply embody theexamples provided herein.

[0190] The invention also includes a kit for identifying a potentialligand of a membrane protein. The kit comprises a lipoparticlecomprising a membrane protein. The kit includes a kit where thelipoparticle is attached to a surface and further includes where alipoparticle is provided separately from the surface, which is alsoprovided in the kit. The kit further comprises a test ligand, or aplurality of such ligands, such as, but not limited to, a library oftest ligands to be assessed for their ability to specifically bind withthe membrane protein present in the lipoparticle.

[0191] The kit further comprises an applicator, where the applicator canbe used to attach the lipoparticle to the surface and/or to apply thetest ligand to the surface such that the test ligand can contact andbind with the lipoparticle bound to the surface.

[0192] The invention includes a kit for identifying a compound thataffects binding between a ligand and a membrane protein receptor. Thekit comprises a lipoparticle comprising a membrane protein and a surfaceto which the lipoparticle can be attached. The kit comprises where thelipoparticle and surface are provided separately or where thelipoparticle is provided already attached to the surface.

[0193] In one aspect, the surface includes a wide variety of sensorsurfaces, such as, but not limited to, a wide plethora of biosensorchips that are known in the art or to be developed in the future.

[0194] Further, as more fully set forth elsewhere herein, the membraneprotein encompasses a wide plethora of membrane spanning proteins thatspan the lipid bilayer at least once.

[0195] Moreover, the kit comprises an applicator and an instructionalmaterial for the use of the kit. These instructions simply embody theexamples provided herein.

[0196] One skilled in the art would appreciate, based upon thedisclosure provided herein, that the invention encompasses a kit wherethe lipoparticle is provided physically separated from a ligand andwhere the ligand is already bound with the lipoparticle. Similarly, theinvention encompasses a kit where the lipoparticle is providedphysically separated from the surface, as well as a kit where thelipoparticle is provided attached to the surface. Further, the skilledartisan would understand that the invention encompasses a kit with allpossible permutations such that the ligand can be bound with thelipoparticle which is, in turn, attached to the surface, or each isprovided separately, or any permutation thereof.

[0197] As more fully set forth elsewhere herein, the kit comprises awide plethora of membrane spanning proteins, lipoparticles, andsurfaces, and combinations thereof. Moreover, the kit comprises anapplicator and an instructional material for the use of the kit. Theseinstructions simply embody the examples provided herein.

[0198] Definitions

[0199] Certain terminology is used herein as follows.

[0200] The articles “a” and “an” are used herein to refer to one or tomore than one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

[0201] By the term “applicator” as the term is used herein, is meant anydevice including, but not limited to, a hypodermic syringe, a pipette,and the like, for attaching a lipoparticle and/or composition of theinvention to a surface, including a sensor surface. Further, theapplicator can be used to contact a ligand and/or a test compound with alipoparticle.

[0202] The term “enveloped virus vector” means an enveloped virus-likeparticle comprising at least one cellular virus receptor proteincontained within the envelope of the particle, wherein the envelopedvirus vector is capable of fusing with a target cell which comprises aviral envelope protein to which the cellular virus receptor protein iscognate.

[0203] The term “enveloped virus-like particle” means a composition ofmatter comprising a replication-competent or replication-incompetentvirus surrounded by a lipid-containing virus envelope and at least oneof a viral envelope protein and a cellular virus receptor protein,wherein the virus-like particle is incapable of replication in theabsence of a cell.

[0204] The term “recombinant enveloped virus vector” means an envelopedvirus vector comprising a nucleic acid which has been manipulated by anyrecombinant nucleic acid protocol known in the art (see, e.g. Sambrook,et al., 1989, In: Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, New York).

[0205] The term “enveloped virus” means a virus comprising an envelope.

[0206] The terms “envelope” and “viral envelope” mean thelipid-containing or lipoprotein-containing membrane which surrounds thevirion of an enveloped virus or at least one component of such a virion.

[0207] The term “cellular virus receptor protein” means a protein which,in a normal cell, is encoded by the cell and, at least under certainconditions, is associated with the outer surface of the membrane of thecell, wherein the protein is capable of specifically interacting with aviral envelope protein of an enveloped virus to facilitate attachment ofthe virus to the cell. A cellular virus receptor protein is functionalif it is located in the outer membrane of the cell and is oriented suchthat the portion of the cellular virus receptor protein which is capableof interacting with a viral envelope protein contacts the extracellularmedium. A cellular virus receptor protein is also functional if it islocated in the envelope of an enveloped virus or a virus vector havingan envelope and it is oriented such that the portion of the cellularvirus receptor protein which is capable of interacting with a viralenvelope protein contacts the medium in which the virus or virus vectoris suspended. A cellular virus receptor protein may be a full-lengthprotein, as encoded by a normal cell, or may be a fragment thereof.

[0208] The term, “cellular,” as it is used to refer to a virus receptorprotein, means that the virus receptor protein is normally encoded bythe cell and not viral DNA. However, the term also applies to a proteinexpressed by a recombinant virus wherein a cellular nucleic acidencoding the receptor protein has been inserted into the genome of therecombinant virus for expression therefrom. Furthermore, the term alsoapplies when the protein is provided to a virus or a virus vector in theform of a protein or a peptide.

[0209] The term “cellular chemokine receptor” means a protein, which,when expressed by a cell which normally encodes the chemokine receptor,is capable of interacting with a chemokine in the extracellular medium.By way of example, CCR5 is a chemokine receptor which, when expressed bya cell which naturally encodes CCR5, is capable of interacting withRANTES, MIP-1alpha, AND MIP-1beta.

[0210] The term “viral envelope protein” means a protein which, in anormal enveloped virus, is encoded by the genome of the virus and isassociated with the envelope of the virus, wherein the protein iscapable of specifically interacting with a cognate cellular virusreceptor protein to facilitate attachment of the virus to a cell. Viralenvelope proteins include, but are not limited to, glycoproteins. Aviral envelope protein is functional if it is located in the envelope ofan enveloped virus or a virus vector and is oriented such that theportion of the viral envelope protein which is capable of interactingwith a cognate cellular virus receptor protein contacts the medium inwhich the virus or virus vector is suspended. A viral envelope proteinis also functional if it is located in the outer membrane of a cell andis oriented such that the portion of the viral envelope protein which iscapable of interacting with a cognate cellular virus receptor proteincontacts the extracellular medium. A functional viral envelope proteinmay be a full-length protein, as synthesized during normal replicationof a virus, or it may be a fragment thereof.

[0211] The term “retroviral envelope protein” means a viral envelopeprotein of a retrovirus.

[0212] A cellular virus receptor protein is “cognate” to a viralenvelope protein if the cellular virus receptor protein is capable ofinteracting with the viral envelope protein and facilitating fusion ofthe membrane or envelope with which it is associated and the membrane orenvelope with which the viral envelope protein is associated.

[0213] A cell and an enveloped virus or enveloped virus vector are“capable of fusing” if a protein of the cell and a protein of the virusor vector are capable of specifically interacting and facilitating thefusion of a membrane of the cell and the envelope of the virus orvector, whereby the contents of the envelope of the virus or vector andthe contents of the membrane of the cell are combined.

[0214] The term “cell” means any type of living cell. Cells of bothunicellular and multicellular organisms are included. Cells ofmulticellular organisms are preferred, and cells of animals are morepreferred. Cells of vertebrates are still more preferred, and cells ofmammals are even more preferred. Most preferably, the cells are humancells.

[0215] The term “virus-infected cell” means a cell which has beeninfected by a virus including, but not limited to, an enveloped virus,and which comprises a viral protein including, but not limited to, aviral envelope protein in its outer membrane.

[0216] The term “producer cell” means a cell in which an enveloped virusor an enveloped virus vector of the invention can be generated.

[0217] The term “target cell” means a cell with which fusion with anenveloped virus or an enveloped virus vector of the invention isdesired. A target cell corresponding to the enveloped virus vector ofthe invention comprises a functional viral envelope protein to which thecellular virus receptor protein of the enveloped virus vector iscognate.

[0218] As used herein, an “instructional material” includes apublication, a recording, a diagram, or any other medium of expressionwhich can be used to communicate the usefulness of the lipoparticleand/or composition of the invention in the kit for assessing proteinbinding, identifying ligands for a membrane protein, identifying acompound that affects a ligand binding with its cognate membranereceptor protein, and the like, as more fully recited elsewhere herein.Optionally, or alternately, the instructional material may describe oneor more methods of using a lipoparticle of the invention. Theinstructional material of the kit of the invention may, for example, beaffixed to a container which contains the lipoparticle and/orcomposition of the invention or be shipped together with a containerwhich contains the lipoparticle and/or composition. Alternatively, theinstructional material may be shipped separately from the container withthe intention that the instructional material and the compound be usedcooperatively by the recipient.

[0219] The term “host cell” means a cell which is susceptible toinfection by an enveloped virus.

[0220] The term “test cell” means a cell which comprises at least one ofa viral envelope protein, a cellular virus receptor protein, and a testprotein.

[0221] The term “control cell” means a cell used in conjunction with atest cell, which is of the same cell type as the test cell, and whichdoes not comprise a viral envelope protein, a cellular virus receptorprotein, or a test protein.

[0222] The term “test protein” means an unknown protein which may be aviral envelope protein, a cellular virus receptor protein, or neither ofthese.

[0223] The term “competent portion of the genome of an enveloped virus”means the portion of the genome of the enveloped virus which, whenexpressed in a cell, results in formation of at least one virus-likeparticle.

[0224] The term “host range” describes an organism or a group oforganisms, the cells of which are susceptible to infection by anenveloped virus.

[0225] The term “tissue tropism” describes a tissue or a group oftissues of an organism, the cells of which are susceptible to infectionby an enveloped virus.

[0226] The term “tropism determinant” means a component of an outermembrane of a cell or a component of an envelope of a virus, whichcomponent is involved in infection of the cell by the virus or iscapable of affecting a second component which is involved in infectionof the cell by the virus. Non-limiting examples of tropism determinantsinclude a cellular virus receptor protein, a viral envelope protein, aprotein which interacts with a cellular virus receptor protein, and aprotein which interacts with a viral envelope protein.

[0227] The term “intracellular antagonist of HIV” means a composition ofmatter which, when provided to the interior of a cell infected with HIV,is capable of interfering with maintenance or replication of HIV or withintegration of the HIV genome into the host cell genome.

[0228] The term “antiviral agent” means a composition of matter which,when delivered to a cell, is capable of preventing replication of avirus in the cell, preventing infection of the cell by a virus, orreversing a physiological effect of infection of the cell by a virus.Antiviral agents are well known and described in the literature. By wayof example, AZT (zidovudine, Retrovir® Glaxo Wellcome Inc., ResearchTriangle Park, N.C.) is an antiviral agent which is believed to preventreplication of HIV in human cells.

[0229] The term “imaging agent” means a composition of matter which,when provided to a cell, facilitates detection of the cell. Numerousimaging agents are known and described in the literature. By way ofexample, enzymes, such as beta-galactosidase, which are capable ofcatalyzing a reaction involving a chromogenic substrate may be providedto a cell. When the chromogenic substrate is reacted with the enzyme,the cell is detectable. In this sense, the enzyme is considered hereinto be an imaging agent. Further by way of example, compounds, thepresence of which may be directly detected, may be provided to a cell,such as compounds which emit gamma radiation or which fluoresce, whichmay be detected using an appropriate detection apparatus.

[0230] The term “antisense nucleic acid” means a nucleic acid polymer,at least a portion of which is complementary to a nucleic acid which ispresent in a cell.

[0231] “Antisense” refers particularly to the nucleic acid sequence ofthe non-coding strand of a double stranded DNA molecule encoding aprotein, or to a sequence which is substantially homologous to thenon-coding strand. As defined herein, an antisense sequence iscomplementary to the sequence of a double stranded DNA molecule encodinga protein. It is not necessary that the antisense sequence becomplementary solely to the coding portion of the coding strand of theDNA molecule. The antisense sequence may be complementary to regulatorysequences specified on the coding strand of a DNA molecule encoding aprotein, which regulatory sequences control expression of the codingsequences.

[0232] “Complementary” as used herein refers to the broad concept ofsubunit sequence complementarity between two nucleic acids, e.g., twoDNA molecules. When a nucleotide position in both of the molecules isoccupied by nucleotides normally capable of base pairing with eachother, then the nucleic acids are considered to be complementary to eachother at this position. Thus, two nucleic acids are complementary toeach other when a substantial number (at least 50%) of correspondingpositions in each of the molecules are occupied by nucleotides whichnormally base pair with each other (e.g., A:T and G:C nucleotide pairs).

[0233] The term “cytotoxic compound” means a composition of matterwhich, when provided to a cell, is capable of killing the cell.

[0234] The term “library” means a plurality of nucleic-acid-containingvectors.

[0235] “A multiple membrane spanning protein,” as the term is usedherein, is a polypeptide that spans the cell membrane at least twice.That is, the peptide is typically present in a cell membrane where itspans the lipid bilayer at least twice.

[0236] The term “non-human animal model of a human disease or disorder”means a non-human animal which has been rendered susceptible toinfection by a human pathogenic enveloped virus and which, when soinfected, exhibits a physiological condition which is analogous to asymptom exhibited by a human infected with the same virus. The term alsomeans a non-human animal which is susceptible to infection by anon-human pathogenic enveloped virus. When the non-human animal isinfected with the non-human pathogenic enveloped virus, the animalexhibits a pathology which is similar to the pathology of a humaninfected with the corresponding human pathogenic enveloped virus. By wayof example, certain known species of monkeys are susceptible toinfection by SIV, giving rise to a disease which is similar to that inhumans infected with HIV.

[0237] The term “staged delivery of enveloped virus vectors” meanssequential delivery to an organism of a plurality of enveloped virusvectors of the invention, wherein a first enveloped virus vector iscapable of fusing with a cell of the organism, and wherein delivery ofthe first enveloped virus vector renders the cell susceptible to fusionwith a second enveloped virus vector, which is subsequently delivered tothe cell.

[0238] The term “pharmaceutically-acceptable carrier” means a chemicalcomposition with which an enveloped virus vector of the invention may becombined for administering the vector to an animal, preferably to ahuman.

[0239] The invention is now described with reference to the followingexamples. These examples are provided for the purpose of illustrationonly and the invention should in no way be construed as being limited tothese examples but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

EXAMPLE 1 An Enveloped Virus Vector Comprising CD4 and a ChemokineReceptor: A Gene Delivery Vector that Fuses with HIV- or SIV-InfectedCells.

[0240] The delivery to HIV- and SIV-infected cells of a gene encodingluciferase protein using the enveloped virus vector of the invention isdescribed. The results presented herein exemplify a novel method todeliver antiviral genes directly to an HIV-1-infected cell in vivo andprovides a novel treatment strategy to complement existing antiviraltherapies. In addition, the method can be used to deliver a component ofan enveloped virus vector to an HIV-infected cell. Furthermore, bysubstituting a viral envelope protein and a cellular virus receptorprotein corresponding to any other enveloped virus, the methodsexemplified herein may be applied to make and use an enveloped virusvector capable of fusing with cells infected with that other envelopedvirus.

[0241] Co-expression of CD4 and an appropriate chemokine receptor issufficient to render human and nonhuman cells susceptible to infectionby HIV or SIV (analogous to FIG. 1, Panel A). In the results describedherein, co-expression of CD4 and a chemokine receptor in the envelope ofan enveloped virus vector was demonstrated to facilitate fusion of theenveloped virus vector with a cell infected with HIV or SIV (analogousto FIG. 1, Panel B).

[0242] An illustration of an aspect of the invention is depicted inFIG. 1. FIG. 1, Panel A is an illustration which depicts entry of HIV orSIV into a cell susceptible to infection by either virus. The circularentity having knobby projections represents the viral envelope of an HIVvirion or an SIV virion, wherein the knobby projections represent viralenvelope proteins. The pair of parallel lines which define a portion ofa rectangle having rounded corners represents a portion of the membraneof a cell. The concave side of the cell membrane represents the interiorface of the cell membrane, and the convex side of the cell membranerepresents the exterior of the cell. CD4 embedded in the cell membraneis represented by a black entity which spans the cell membrane and has asingle semi-circular end. A cytokine receptor protein is represented bya series of irregular lines, some of which span the cell membrane.Interaction among at least one viral envelope protein, CD4, and at leastone cytokine receptor leads to fusion of the viral envelope and the cellmembrane, whereby the contents of the viral envelope are delivered tothe interior of the cell.

[0243]FIG. 1, Panel B is an illustration which depicts targeting ofcells infected with HIV or SIV using an enveloped virus vector of theinvention. In this panel, the representations are the same as those usedin Panel A. However, in Panel B, the viral envelope proteins are presentin the cell membrane, representing a cell which is infected with HIV orSIV. CD4 and a cytokine receptor are present in the envelope of theenveloped virus vector. Analogously to the situation depicted in PanelA, but in the opposite orientation, interaction among at least one viralenvelope protein, CD4, and at least one chemokine receptor leads tofusion of the envelope of the enveloped virus vector and the cellmembrane, whereby the contents of the envelope of the enveloped virusvector are delivered to the interior of the cell.

[0244] The materials and methods used in the experiments presented inExample 1 are now described.

[0245] The enveloped virus vector used in Example 1 comprised one ormore of CD4 and a chemokine receptor. The enveloped virus vector wasgenerated by providing to a producer cell line the HIV-1 provirus,NL-R⁻E⁻luc, which comprises a competent portion of the HIV-1 genome.NL-R⁻E⁻luc (Connor et al., 1995, Virology 206:935-944) is deficient forenv and nef; two viral genes, the expression of which is known to reducethe amount of CD4 on the plasma membrane (Crise et al., 1990, J. Virol.64:5585-5593; Jabbar et al., 1990, J. Virol. 64:6297-6304; Garcia etal., 1991, Nature 350:508-511; Mariani et al., 1993, Proc. Natl. Acad.Sci. USA 90:5549-5553; Benson et al., 1993, J. Exp. Med. 177:1561-1566;Aiken et al., 1994, Cell 76:853-864). NL-R⁻E⁻luc also comprises aluciferase reporter gene that facilitates detection and quantitation ofthe fusion of the enveloped virus vector with a target cell.

[0246] The enveloped virus vectors described in Example 1 were generatedby transient cotransfection of approximately 5×10⁶ producer cells usingone or more of four plasmids. An enveloped virus vector comprising CD4and a chemokine receptor was generated using 10 micrograms of plasmidpNL-R⁻E⁻luc (which comprises the NL-R⁻E⁻luc provirus; Connor et al.,1995, Virology 206:935-944), 10 micrograms of plasmid pT4 (whichcomprises a gene encoding CD4; Littman et al., 1985, Cell 40:237-246),and 10 micrograms of either plasmid pLESTR/cDNA3 (which comprises a geneencoding CXCR4; Berson et al., 1996, J. Virol. 70:6288-6295) or plasmidpCKR5/cDNA3 (which comprises a gene encoding CCR5; Doranz et al., 1996,Cell 85:1149-1158). Other enveloped virus vectors were generated whereinthe plasmid encoding CD4, the plasmid encoding a chemokine receptor, orboth were omitted, and wherein an equivalent quantity of plasmid pcDNA3(which is commercially available, e.g. from Invitrogen, Carlsbad,Calif.) was substituted for the omitted plasmid(s), so that a total of30 micrograms of plasmid DNA per transfection was used in each case.Forty-eight hours post-transfection, transfected producer cells inculture were harvested, and enveloped virus vector was collected bypassing the culture medium through a membrane having a pore size of 0.22micrometer to remove producer cells. Preparations of enveloped virusvector were divided into aliquots, and the aliquots were stored at

[0247] −80° C. Each preparation of enveloped virus vector was quantifiedusing a commercial HIV-1 p24 assay (Dupont; Wilmington, Del.).

[0248] The producer cells used in Example 1 were QT6 cells, which arequail cells that lack both CD4 and chemokine receptors (Moscovici etal., 1977, Cell 11:95-103; Doranz, 1996, Cell 85:1149-1158). Envelopedvirus vector preparations were normalized to achieve equivalent p24levels among the preparations.

[0249] The ability of the enveloped virus vector to fuse with CEMx174target cells which were chronically infected with HIV or SIV wasassessed by contacting the vector with target cells as follows. Infectedor non-infected target cells were transferred to 24-well plates. Eachwell contained approximately 5×10⁴ target cells and an amount of anenveloped virus vector preparation corresponding to 10 nanograms of p24for the preparation. The target cells and the enveloped virus vectorwere incubated overnight. The following day, 0.5 milliliter of freshmedium (comprising RPMI-1640 medium supplemented with 10% (v/v) fetalbovine serum, 100 units per milliliter penicillin, 100 micrograms permilliliter streptomycin, and 2 millimolar L-glutamine) was added to eachwell. Four days postinfection, cell cultures were harvested, pelleted,washed twice with phosphate-buffered saline (PBS), and the cells werelysed using 150 microliters of luciferase lysis buffer (Promega Corp.;Madison, Wis.). The amount of luciferase activity present in 20microliters of lysate was assessed using commercially availablereagents, according to the manufacturer's instructions (Promega Corp.;Madison, Wis.). Experiments were repeated at least twice in duplicate,and reported values represent the average of duplicate samples±thestandard error of the mean (SEM).

[0250] Expression of SIV Viral Envelope Proteins

[0251] Target cells were chronically infected with SIVmac239 orSIVmac239/MT. SIVmac239/MT is a variant of SIVmac239 in which thecytoplasmic portion of the transmembrane envelope protein is modified bya tyrosine-to-cysteine substitution at amino acid position 721 and apremature stop codon is present at amino acid position 734. It has beendemonstrated that another SIV viral envelope protein containing thesesubstitutions exhibits markedly enhanced levels of expression on theplasma membrane of the cells infected therewith (Labranche et al., 1995,J. Virol. 69:5217-5227; Sauter et al., 1996, J. Cell Biol. 132:795-811).SIVmac239/MT was constructed by cloning the 475 nucleotide Nhe1-Bgl2fragment from pCPenv into the corresponding sites of pVP-2. To recoverinfectious virus, the resulting plasmid (p3'239/MT) and pVP-1 weredigested with Sph1, ligated, and transfected into target cells.Supernatants were harvested, clarified by centrifugation for fiveminutes at 2,000× g, and SIVmac239/MT was obtained by filtration using amembrane having a pore diameter of 0.22 micrometer. Preparations ofvirus were divided into aliquots, and the aliquots were stored at −80°C.

[0252] The quantity of viral envelope protein α-SIV gp12O expressed onthe surface of cells chronically infected with SIVmac239 or SIVmac239/MTwas estimated by flow cytometry using a FACScan analyzer (BectonDickenson). Pelleted cells were resuspended in ice-cold staining bufferwhich comprised PBS, 0.1% (w/v) bovine serum albumin, 0.02% (w/v) sodiumazide, and mAb101.1, a monoclonal antibody which specifically recognizesα-SIV gp12O (Labranche et al., 1995, J. Virol. 69:5217-5227; Sauter etal., 1996, J. Cell Biol. 132:795-811). Following suspension, cells wereincubated for 30 minutes at 4° C. with fluoresceinisothiocyanate-conjugated F(ab′)₂ goat anti-mouse immunoglobulin G.Cells were washed with PBS and fixed in 4% (v/v) paraformaldehyde priorto FACS analysis. Staining results did not appear to beepitope-dependent, since similar staining patterns were obtained usingother primary antibodies (Labranche et al., 1995, J. Virol.69:5217-5227; Sauter et al., 1996, J. Cell Biol. 132:795-811) directedagainst SIV gp41 (mAb 43.1) or gp120 (mAb 7D3 and mAb 5B11).

[0253] Enveloped virus vectors were preincubated for two hours at 37° C.with either neutralizing monoclonal antibody α-CD4#19 (Endres et al.,1996, Cell 87:745-756) or antibody DL11, an isotyped matched control.Approximately 5×10⁴ CEMx174 cells which were chronically infected witheither HIV or SIV were added to each well of a 24-well plate. Anenveloped virus vector which had been preincubated with one of the twoantibodies was added to each well in an amount corresponding to 10nanograms of p24 for the preparation, and the final concentration ofantibody was maintained at 15 micrograms per milliliter in each well.The cells, vectors, and antibodies were incubated for three to four daysat 37° C. Following incubation, cells were harvested, washed with PBS,lysed in 150 microliters of luciferase lysis buffer (Promega Corp.,Madison Wis.), and the amount of luciferase activity in 20 microlitersof lysate was assessed. In selected wells, the enveloped virus vectorwas not preincubated with α-CD4#19, and α-CD4#19 was not added to thewell until sixteen hours after the cells and vector were mixed. Reportedvalues represent the average of duplicate samples±SEM. Experiments wererepeated twice in duplicate, and similar results were obtained in eachexperiment.

[0254] Monocyte-derived macrophages (MDM) were isolated from peripheralblood mononuclear cells of healthy seronegative donors and maintained inmacrophage media as previously described (Collman et al., 1989, J. Exp.Med. 170:1149-1163). Approximately 4×10⁵ MDM cells were cultured foreight days in individual wells of 24-well plastic tissue culture platesprior to infection of the MDM with HIV-1/89.6. No later than ten dayspostinfection, p24 antigen could be detected in MDM cultures which wereinfected with HIV, and extensive syncytia were present. Ten dayspostinfection, MDM were contacted with an enveloped virus vector.Following an additional four days of incubation at 37° C., luciferaseactivity was assessed in the MDM as described herein. Reportedluciferase activity values represent the average of duplicate samples+SEM.

[0255] The results obtained in the experiments presented in Example 1are now described.

[0256] Enveloped virus vectors comprising CD4 and a chemokine receptorwere evaluated for their ability to transduce HIV- and SIV-infectedtarget cells. As depicted in FIG. 2, Panel A, an enveloped virus vectorcomprising CD4 and a particular chemokine receptor was able to transduceHIV- or SIV-infected cells in a manner that corresponded to whether thecellular virus receptor protein(s) were cognate to the viral envelopeprotein(s) which were expressed on the cells. Accordingly, an envelopedvirus vector comprising CD4 and CXCR4 was able to fuse with CEMx 174cells which had been infected with HIV-1/IIIB or HIV-1/89.6, but was notable to fuse with cells which had been infected with SIVmac239. Anenveloped virus vector comprising CD4 and CCR5 was able to fuse withCEMx174 cells which had been infected with SIVmac239 or HIV-1/89.6, butwas not able to fuse with cells which had been infected with HIV-1/IIB.None of the enveloped virus vectors comprising a cellular virus receptorprotein were able to fuse with non-infected cells.

[0257] The amount of viral envelope protein expressed on infected CEMx174 cells significantly influenced the efficiency of fusion withenveloped virus vectors. Cells which were chronically infected withSIVmac239/MT, an engineered variant of SIVmac239, exhibitedapproximately 10-fold higher surface envelope protein expression, asassessed by FACS using gp120- and gp41-specific monoclonal antibodies(FIG. 2, Panel B). Cells infected with SIVmac239/MT also displayedmarkedly increased susceptibility to fusion with enveloped virus vectorswhich comprised CD4 and CCR5 (FIG. 2, Panel A).

[0258] These studies further demonstrate that an enveloped virus vectorcomprising a cellular virus receptor protein which is cognate to a viralenvelope protein of HIV can be used to deliver the vector specificallyto HIV-infected cells.

[0259] Effect of CD4 Availability

[0260] Studies were performed to evaluate the dependence upon theavailability of CD4 of the ability of the vector to fuse with CEMx174cells using enveloped virus vectors comprising CD4 and either CXCR4 orCCR5. As depicted in FIG. 3, Panel A, addition to the infection mixtureof α-CD4#19, a monoclonal antibody specific for CD4 which has beendemonstrated to block HIV and SIV infection of cells (Endres et al.,1996, Cell 87:745-756), completely inhibited fusion of HIV- orSIV-infected CEMx174 cells with enveloped virus vectors comprising CD4and either CXCR4 or CCR5. Remarkably, α-CD4#19 minimally inhibitedfusion of enveloped virus vectors comprising CD4 and CXCR4 with CEMx174cells which had chronically infected by HIV-2/VCP, an HIV-2 variantdemonstrated to utilize CXCR4 as a receptor in the absence of CD4(Endres et al., 1996, Cell 87:745-756). Taken together, these resultsindicate that fusion of enveloped virus vectors with cells correlatedwith the amount of viral envelope protein on the cell surface andreflected the CD4 dependence of the virus with which the cells wereinfected.

[0261] The ability of enveloped virus vectors comprising CD4 and eitherCXCR4 or CCR5 to target HIV- or SIV-infected cells was consistent with asingle round of entry. However, it was theoretically possible thatfollowing fusion of the vector with the cell, the NL-R⁻E⁻luc recombinantretrovirus vector replicated and was subsequently packaged with HIV orSIV envelope glycoproteins expressed by the target cell to forminfectious particles. If formed, these infectious particles couldpotentially infect CD4+cells in a subsequent round of entry. To confirmthat infection of target cells by such infectious particles did notoccur, expression of the gag gene in the target cells was determined andthe presence on the surface of the target cells of CD4⁺ was assessed byfluorescence activated cell sorting (FACS). One hundred percent oftarget cells were infected, as determined by detection of intracellulargag gene products (Labranche et al., 1995, J. Virol. 69:5217-5227;Sauter et al., 1996, J. Cell Biol. 132:795-811). Furthermore, targetcells expressed no detectable surface CD4, as assessed by FACS.Therefore these cells were resistant to superinfection by any infectiousparticles that might have been formed. To further confirm that infectionof target cells by infectious particles did not occur, α-CD4#19 antibodywas added to the infection mixture sixteen hours after the initiation ofincubation of the target cells with a recombinant retrovirus vector. Thepurpose of adding the antibody was to inhibit any superinfection whichmight involve CD4. As demonstrated by the results depicted in FIG. 3,Panel B, inhibition of fusion of the enveloped virus vector with thecells was apparent when α-CD4#19 antibody was added concurrently withthe addition of vector, but not when α-CD4#19 antibody was added sixteenhours after the initiation of incubation of the cells with the vector.Taken together, these results indicate that a second round of infectiondid not occur in the target cells.

[0262] Fusion of the Enveloped Virus Vector with Monocyte-DerivedMacrophages

[0263] In order to demonstrate that the results obtained using CEMx 174cells are equally applicable to a primary cell type, the ability of anenveloped virus vector comprising CD4 and either CXCR4 or CCR5 to fusewith monocyte-derived macrophages which had been acutely infected withHIV was examined. An enveloped virus vector comprising CD4 and CXCR4 wasable to fuse with MDM which had been acutely infected with HIV-1/89.6(FIG. 4) and was unable to fuse with non-infected MDM. Furthermore, anenveloped virus vector comprising CD4, but not comprising CXCR4, wasunable to fuse with MDM that had been acutely infected with HIV-1/89.6.

[0264] HIV-derived enveloped virus vectors offer a distinct advantageover enveloped virus vectors derived from avian or murine viruses withrespect to targeting post-mitotic cells such as macrophages (Naldini etal., 1996, Science 272:263-267). Transduction of HIV-1-infected MDM maybe enhanced if the enveloped virus vector further comprises Vpr protein(Connor et al., 1995, Virology 206:935-944; Naldini et al., 1996,Science 272:263-267).

[0265] The results presented in this Example demonstrate that anenveloped virus vector comprising CD4 and a cytokine receptor is aneffective vehicle for delivering vector components, such as genes,directly and specifically to HIV- and SIV-infected cells, includingnon-dividing, post-mitotic cells. Furthermore, these results demonstratethat fusion resulting from the interaction between the HIV and SIVenvelope proteins and their cognate cellular virus receptor proteins isnot dependent upon which protein is borne by the cell and which is borneby the vectors.

[0266] An enveloped virus vector comprising a cellular virus receptorprotein can be used to deliver a vector component to HIV- andSIV-infected cells. In addition to providing a potential therapeuticstrategy to target reservoirs of HIV-infected cells in patients,enveloped virus vectors comprising CD4 and a cytokine receptor alsoprovide a convenient means for screening compounds for their ability tointerfere with interactions between a cellular virus receptor proteinand a viral envelope protein to which it is cognate. Because theorientation of the cellular virus receptor protein and the viralenvelope protein have been reversed, this system permits identificationof compounds which exert their inhibitory effects by true stericinterference. Thus, compounds which exert their inhibitory effects bysteric interference may be distinguished from compounds which, forexample, induce receptor internalization or desensitization. Inaddition, reversing the orientation of interactions between acell-encoded cellular virus receptor protein and a virus-encoded viralenvelope protein to which it is cognate may be applied analogously withrespect to any enveloped virus and can be used as a general approach toscreen mammalian cells for cellular virus receptor proteins.

EXAMPLE 2 Efficient Infection Mediated by an Enveloped Virus VectorComprising a Cellular Virus Receptor Protein.

[0267] Methods which were used to make and use enveloped virus vectorscomprising a cellular virus receptor protein are described in thisExample. As described herein, these vectors were able to deliver a geneto the interior of a cell comprising a viral envelope protein to whichthe cellular virus receptor protein is cognate.

[0268] Many host cell surface proteins, including cellular virusreceptor proteins, are capable of being incorporated into the envelopeof an enveloped virus. To assess the functional significance of thesecell-encoded proteins, murine leukemia virus vectors were produced,wherein the viruses comprised either the cellular virus receptor proteinfor Rous sarcoma virus (hereinafter, “RSV”) or the cellular virusreceptor protein for ecotropic murine leukemia virus (hereinafter,“MLV”). These receptor-pseudo-typed murine leukemia viruses(hereinafter, “RPMLV”; an embodiment of the enveloped virus vector ofthe invention) efficiently infected cells which expressed a viralenvelope protein to which the cellular virus receptor protein of theRPMLV was cognate.

[0269] RPMLV were constructed which comprised a cellular virus receptorprotein. When the cellular virus receptor protein was Tva, the subgroupA RSV (hereinafter, “RSV(A)”) receptor protein, the RPMLV was designatedMLV(Tva). When the cellular virus receptor protein was MCAT-1, theecotropic MLV receptor, the RPMLV was designated MLV(MCAT). Tvainteracts with EnvA, a viral envelope protein of RSV(A), and cellsexpressing Tva have no apparent requirement for additional factors orco-receptors to mediate infection of the cells by RSV(A) (Bates et al.,1993, Cell 74:1043-1051; Connolly et al., 1994, J. Virol. 68:2760-2764;Gilbert et al., 1994, J. Virol. 68:5623-5628). MCAT-1 is a multiplemembrane spanning amino acid transport protein which has physicalproperties distinct from Tva. Cells expressing MCAT-1 do not appear torequire additional factors or co-receptors to mediate infection of thecells by MLV.

[0270] MLV(Tva) was made by transiently co-transfecting 6×10⁶ 293T cellsusing 15 micrograms of each of three plasmids as described (Soneoka etal., 1995, Nucl. Acids Res. 23:628-633). The three plasmids used forco-transfection were plasmid pCB6 0.95, which encodes the tva gene,plasmid pHit60, which encodes MLV gag-pol, and plasmid pHit111, an MLVpacking genome which encodes a β-galactosidase reporter gene. Thirty-sixhours post transfection, the medium comprising the transfected cells washarvested and clarified by centrifugation at 2,300× g to yield culturesupernatant. The culture supernatant was divided into aliquots, and thealiquots were stored at −80° C. until they were used for infectionassays.

[0271] Incorporation of Tva protein into the envelope of RPMLV wasassessed by equilibrium density gradient centrifugation analysis oftransiently-produced RPMLV particles. Culture supernatant wascentrifuged for eighty-five minutes at 28,000 rotations per minute in anSW 28 rotor in a centrifuge tube containing a 20% (w/v) sucrose solutionlayered on top of a 60% (w/v) sucrose solution. Following thiscentrifugation, a virion suspension was retrieved from the portion ofthe centrifuge tube which contained the 20%/60% (w/v) sucrose interface.The virions were diluted and the suspension was centrifuged for fiftyminutes at 41,000 rotations per minute in an SW41 rotor in a centrifugetube containing a 20% (w/v) sucrose solution layered on top of a 60%(w/v) sucrose solution. Following this centrifugation, a second virionsuspension was retrieved from the portion of the centrifuge tube whichcontained the 20%/60% (w/v) sucrose interface. The second virionsuspension was diluted three-fold with PBS and layered on top of a15%-45% (w/v) sucrose gradient in a gradient centrifuge tube. Thegradient centrifuge tube was centrifuged for sixteen hours in an SW 41rotor at 35,000 rotations per minute. Following this centrifugation, onemilliliter fractions were collected from the gradient centrifuge tube.Each fraction was centrifuged for ten minutes at 55,000 rotations perminute in an SW55 rotor in a centrifuge tube containing a 20% sucrosesolution layered on top of a 60% (w/v) sucrose solution. A third virionsuspension was retrieved from the portion of the centrifuge tube whichcontained the 20%/60% (w/v) sucrose interface. The virions in the thirdvirion suspension were then lysed using RIPA buffer. RIPA buffercomprises 140 millimolar NaCl, 10 millimolar Tris buffer adjusted to pH8.0, 5 millimolar EDTA, 1% (w/v) sodium deoxycholate, 1% (v/v) TritonX-100, and 0.1% (w/v) sodium dodecyl sulfate. Suspension of virions inRIPA buffer is sufficient to lyse virions and solubilize virionproteins. The proteins in the lysed virion suspension were separated ona 12.5% (w/v) SDS-PAGE gel, transferred to nitrocellulose, and subjectedto Western blot analysis using either anti-Gag antibody (Rong et al.,1997, J. Virol. 71:3458-3465) or anti-Tva antibody (Rong et al., 1995,J. Virol. 69:4847-4853). Western blot analysis of the SDS-PAGE-separatedproteins of the lysed virion suspension revealed that MLV Gag proteinsp30 and Pr65 co-sedimented with the multiple bands characteristic of Tva(FIG. 5). Furthermore, virions comprising Tva were recovered fromsucrose gradient fractions corresponding to the density of intact MLVvirions under these conditions (Young et al., 1990, Science250:1421-1423; Jones et al., 1990, J. Virol. 64:2265-2279). In parallelexperiments in which pHit60 was omitted from the transfection mixture,Tva was not recovered from fractions of the sucrose gradientcorresponding to the density of intact MLV virions. These resultsdemonstrated that Tva was incorporated into intact virions to produceMLV(Tva).

[0272] The ability of MLV(Tva) to infect cells was evaluated using cellschronically infected with RSV(A). Quail QT6 cells were infected withRCAS(A)AP, a replication-competent RSV vector comprising an alkalinephosphatase (AP) reporter gene. After several passages, all QT6 cellsexhibited AP reporter phenotype and appeared to be chronically infected.When cells which had been infected with RCAS(A)AP were exposed toMLV(Tva), fision of the cells with MLV(Tva) was observed. The MLV(Tva)preparation exhibiting a virus titer of 7×10² fusible units permilliliter was used as a stock suspension (Table 1). QT6 cells that hadnot been infected with RCAS(A)AP were not susceptible to fusion withMLV(Tva). These experiments demonstrate that expression of a viralenvelope protein by cells infected with an enveloped virus renders themsusceptible to infection by an enveloped virus vector comprising acognate cellular virus receptor protein. Table 1. Cells expressingdifferent forms of RSV Env protein were infected overnight withMLV(Tva), fixed, and examined for β-galactosidase activity thirty-sixhours post-infection. Virus titer was determined by enumerating cellshaving β-galactosidase activity, and is expressed as fusible units permilliliter (FU/ml). The data shown is representative of multipleexperiments. Virus Titer (FU/ml) 293T Cells QT6 Cells Chronicallyinfected target cells uninfected — 0 RSV(A) Infected — 700 Transfectedtarget cells EnvA 20,000 1,800 EnvC 0 0 EnvA C1⁻ 18 0 EnvA GPI 0 0 EnvAA34[A]Q35 22 3 Mock 0 0

[0273] In another set of experiments, cells transiently expressingvarious RSV viral envelope proteins and mutants thereof were used astargets for fusion with MLV(Tva). In these experiments, a vectorcomprising a nucleic acid which encoded one of the RSV envelopeglycoproteins or mutants listed in Table 1 was used to transientlytransfect 4×10⁵ QT6 cells or 6×10⁵ human 293T cells overnight usingCaPO₄. The quantity of vector used was equivalent to 3 micrograms ofexpression plasmid DNA which comprised the env genes listed in Table 1.Transfected cells were contacted overnight with MLV(Tva) forty-eighthours post-transfection. Two days post-infection, the cells were fixedand stained for β-galactosidase activity. MLV(Tva) efficiently fusedwith both quail and human cells which were transfected with the vectorencoding EnvA protein. Cells subjected to identical procedures, with theexception that no DNA was used to transfect the cells (denoted “Mock” inTable 1) were not susceptible to fusion with MLV(Tva). It has beenreported that Tva binds specifically to subgroup A viral envelopeproteins and does not mediate infection by other subgroups of RSV (Bateset al., 1993, Cell 74:1043-1051; Connolly et al., 1994, J. Virol.68:2760-2764; Gilbert et al., 1994, J. Virol. 68:5623-5628). Consistentwith these reports, transient expression of an RSV subgroup C viralenvelope protein (EnvC) did not render either quail or human cellssusceptible to infection by MLV(Tva).

[0274] Although EnvA is competent to bind receptor in the absence ofprocessing by a host cell, proteolytic cleavage of EnvA is required formaximal fusogenic activity (Freed et al., 1989, J. Virol. 63:4670-4675;McCune et al., 1988, Cell 53:55-67; Perez et al., 1987, J. Virol.61:1609-14). RSV having an envelope comprising a cleavage deficient formof EnvA, EnvA CL⁻, had a fusible titer between four and five orders ofmagnitude lower than wild type RSV. Human 293T cells which weretransfected with a vector encoding EnvA CL⁻ were susceptible to fusionwith MLV(Tva) at a titer roughly three orders of magnitude lower thanthe titer at which 293T cells were transfected with wild type EnvA.

[0275] Viral envelope proteins anchored by aglycosylphosphatidylinositol (GPI) moiety bind to their cognate cellularvirus receptor proteins, but do not mediate fusion of the cell membranewith the viral envelope, and allow only partial mixing of cell membraneand viral envelope lipids (Gilbert et al., 1994, J. Virol. 68:5623-5628;Kemble et al., 1994, Cell 76:383-391; Melikyan et al., 1995, J. CellBiol. 131:679-691; Salzwedel et al., 1993, J. Virol. 67:5279-5288; Weisset al., 1993, J. Virol. 67:7060-7066). Consistent with theseobservations, cells transfected with a vector encoding the GPI anchoredmutant of EnvA (EnvA GPI) were not susceptible to fusion with MLV(Tva).

[0276] Another mutant EnvA protein, EnvA A34[A]Q35, contains aninsertion in the putative fusion peptide of RSV envelope whichdramatically reduces EnvA mediated fusion. Consistent with thisproperty, both quail and human cells which were contacted with a vectorencoding EnvA A34[A]Q35 were susceptible to fusion with MLV(Tva), butthe titer in each cell line was roughly three orders of magnitude lowerthan the titer of the enveloped virus vector comprising wild type EnvA.

[0277] The results of these experiments which assessed thesusceptibility to fusion with MLV(Tva) of human and quail cellstransiently expressing various RSV viral envelope proteins and mutantsthereof are summarized in Table 1 and as follows. The experimentsdemonstrated that the presence on a cell of a viral envelope protein towhich a cellular virus receptor protein is cognate is a criticalrequirement for fusion of the cell with an enveloped virus vectors whichcomprises the cellular virus receptor protein. Fusion of cellscomprising a viral envelope protein with an enveloped virus vectorcomprising a cognate cellular virus receptor protein is at leastsometimes mediated by interaction between the viral envelope protein andthe cognate cellular virus receptor protein.

[0278] Transiently transfected human and quail cells and RSV(A)-infectedquail cells express high levels of EnvA. In order to determine whethercells which express a low level of EnvA would be as efficiently fusedwith MLV(Tva) as cells which express high levels, 3T3EnvA cells, aNIH3T3 cell line which stably expresses EnvA (Gilbert et al., 1994, J.Virol. 68:5623-5628), were employed. Fusion of 3T3EnvA cells withMLV(Tva) was also efficient, the titer ranging from about 2×10³ to about5×10³ FU/ml. Furthermore, the titer of MLV(Tva) using 3T3EnvA cells canbe increased 40-fold to about 2×10⁵ FU/ml by concentrating thesuspension of MLV(Tva) (Burns et al., 1993, Proc. Natl. Acad. Sci.U.S.A. 90:8033-8037). The results of these experiments demonstrate thata high surface density of viral envelope protein on the target cell isnot a prerequisite for efficient fusion of the cell with an envelopedvirus vector comprising a cognate cellular virus receptor protein.

[0279] RPMLV having an envelope comprising Tva*, a nonfunctional mutantof Tva, (hereinafter “MLV(Tva*)”) was produced. The amino acid sequenceof Tva* differs from that of Tva at five amino acid residues. These fiveamino acid sequence differences abrogate the ability of Tva to bind toEnvA in the viral envelope of RSV or to facilitate EnvA-mediatedinfection of cells by RSV. MLV(Tva*) was produced as described in thisExample except that a vector encoding Tva* was used in place of thevector encoding Tva. Western blot analysis of MLV(Tva*) virions whichwere isolated as described herein revealed that Tva* was incorporatedinto MLV virions to approximately the same degree as that to which Tvawas incorporated into MLV virions following transient transfection.MLV(Tva*) was unable to infect 3T3EnvA cells. These results demonstratethat Tva, the RSV cellular virus receptor incorporated into MLV(Tva),was responsible for the infectivity of MLV(Tva).

[0280] Anti-Tva rabbit polyclonal antiserum was raised using standardmethods (see, e.g., Harlow et al., 1988, Antibodies: A LaboratoryManual, Cold Spring Harbor, N.Y.) which antiserum comprised antibodieswhich recognize and bind to Tva protein. MLV(Tva) was incubated withpre-selected dilutions of anti-Tva antiserum at 37° C. for thirtyminutes. Following incubation of the mixture of a particular dilution ofanti-Tva antiserum and MLV(Tva), 3T3EnvA cells were exposed to themixture for four hours at 37° C. Following exposure of the 3T3EnvA cellsto the mixture, the cells were washed twice with phosphate-bufferedsaline (PBS) and cultured in fresh medium comprising the same dilutionof anti-Tva antiserum for forty-eight hours. The 3T3EnvA cells were thenfixed and stained to detect β-galactosidase reporter gene expression.Percent inhibition of fusion was determined by comparing the proportionof cells having β-galactosidase activity to those not having suchactivity in two groups of cells: those treated with MLV(Tva) and thosetreated with MLV(Tva) which had been incubated with anti-Tva antisera.Fusion of 3T3EnvA cells with MLV(Tva) was inhibited in a dose-dependentmanner by incubation of MLV(Tva) with anti-Tva antiserum (FIG. 6, PanelA).

[0281] In another experiment, 3T3EnvA cells were incubated for thirtyminutes at 25° C. in medium comprising a pre-selected concentration ofsTva, a soluble form of Tva RSV cellular virus receptor. Followingincubation, the medium was removed and replaced with a second mediumcomprising the same concentration of sTva and MLV(Tva), and the 3T3EnvAcells were incubated in the second medium for four hours at 37° C. Thecells were then washed twice with PBS and cultured in fresh medium forforty-eight hours. The 3T3EnvA cells were then fixed and stained todetect β-galactosidase reporter gene expression, an indicator of cellfusion with MLV(Tva). Percent inhibition of fusion was determined bycomparing the proportion of cells having β-galactosidase activity tothose not having such activity in two groups of cells: those treatedwith MLV(Tva) and those treated with MLV(Tva) after having beenincubated with sTva. Fusion of 3T3EnvA cells with MLV(Tva) was inhibitedby sTva in a competitive fashion when sTva was present in the mediumbefore and during infection (FIG. 6, Panel B).

[0282] Taken together, the results obtained in experiments involvinginhibition by anti-Tva antisera or by sTva of fusion of 3T3EnvA cellswith MLV(Tva) demonstrate that the capacity of MLV(Tva) to fuse withcells comprising EnvA depends upon the presence in the envelope ofMLV(Tva) of Tva.

[0283] RPMLV exhibits similar specificity and requirements for viralenvelope protein and cellular virus receptor protein as RSV, except thatthe orientation of the proteins during binding and membrane fusion isreversed, the viral envelope protein being on the cell surface and thecellular virus receptor protein being on the surface of the viralparticle. Furthermore, the fusible titers obtained using RPMLV are onlyone or two orders of magnitude lower than titer obtained using RSV orusing Ebola viral envelope protein pseudotypes of MLV. The fact thatinfection of a host cell by RPMLV is efficient demonstrates thatspecific interaction between the viral envelope protein and corecomponents of the virus is not required for uncoating of the virus andinfection of the host cell.

[0284] The cellular MLV receptor, MCAT-1, is an amino acid transportercomprising multiple membrane-spanning domains and is structurallydifferent from Tva (Albritton et al., 1989, Cell 57:659-666; Kim et al.,1991, Nature 352:725-728). A RPMLV comprising MCAT-1 was constructed andwas designated MLV(MCAT).

[0285] MLV(MCAT) was made by cotransfecting 15 micrograms of each ofthree plasmids into 6×10⁶ 293T cells overnight using CaPO₄ as described(Soneoka et al., 1995, Nucl. Acids Res. 23:628-633). The three plasmidsused for co-transfection were plasmid pcDNA3 MCAT-1:Flu3, whichcomprises the gene encoding MCAT-1, plasmid pHit60, and plasmid pHit111.Thirty six hours post transfection, the medium in which the transfectedcells were maintained was harvested and clarified by centrifugation at2,300× g. Incorporation of MCAT-1 into MLV particles was confirmed bypelleting the particles by centrifuging a sample of the supernatant inan SW55 rotor for fifteen minutes at 55,000 rotations per minute in thepresence of 30% (w/v) sucrose. Viral particles in the pellet were lysedusing RIPA buffer, and the proteins therein were separated by 12.5%SDS-PAGE. The proteins which had been separated by PAGE were transferredto nitrocellulose and subjected to Western blot analysis using ananti-HA antibody, 12CA5, which specifically binds to MCAT-1 protein(see, e.g., Pelchen-Matthews et al., 1989, EMBO J. 8:3641-3649). Westernblot analysis indicated that MCAT-1 had been incorporated into MLVparticles. The supernatant was divided into aliquots, and the aliquotswere stored at −80° C. until they were used for infection assays.

[0286] The ability of MLV(MCAT) to infect cells was evaluated using 293Tcells which transiently expressed a fusion-competent form of MLV viralenvelope protein. Titer of fusible units was in the range from about 103to about 104 fusible units per milliliter. No fusion of cells andMLV(MCAT) was observed when mock transfected cells or 293T cellsexpressing either EnvA protein or amphotropic MLV viral envelope proteinwere used. These results demonstrate that MCAT-1 directs fusion of anenveloped virus vector comprising MCAT-1 specifically with cells whichexpress MLV viral envelope protein. Furthermore, the specificityobserved between MLV viral envelope protein and MCAT-1 is retained whenthe viral envelope protein is present in the membrane of a cell andcellular virus receptor protein is present in the envelope of anenveloped virus vector.

[0287] The results presented herein demonstrate that a cell whichcomprises a viral envelope protein are susceptible to fusion with anenveloped virus vector comprising a cognate cellular virus receptorprotein. The results herein also demonstrate that the interactionbetween the cell and the vector is mediated by the interaction betweenthe viral envelope protein and the cognate cellular virus receptorprotein. To the extent that properties of the cell or of the vector orboth do not interfere with the interaction between the viral envelopeprotein and the cognate cellular virus receptor protein, thoseproperties may be varied. These results demonstrate that cellularmembrane receptors can be incorporated into virions and retain theirstructural integrity as measured by functional ability to mediatefusion.

EXAMPLE 3 Use of Virus Particles Comprising Host Cell Surface Protein toAssay Protein-Protein Interaction

[0288] The HIV envelope (Env) protein mediates entry into cells bybinding CD4 and an appropriate coreceptor, which triggers structuralchanges in Env that lead to fusion between the viral and cellularmembranes. The major HIV-1 coreceptors are the seven transmembranedomain chemokine receptors CCR5 and CXCR4. The type of coreceptor usedby a virus strain is an important determinant of viral tropism andpathogenesis, and virus-receptor interactions can e therapeutic targets.However, Envs from many virus strains interact with CXCR4 and CCR5 withlow affinity such that direct study of this important interaction isdifficult if not impossible using standard cell-surface bindingtechniques.

[0289] The data disclosed herein demonstrate a novel approach that makesit possible to study ligand binding to membrane proteins, includingEnv-coreceptor interactions, using a microfluidic device that detectsintramolecular interactions—otherwise known as an optical biosensor.CCR5, CXCR4, and other membrane proteins were incorporated intoretrovirus particles, which were purified and attached to the biosensorsurface. Binding of conformationally sensitive antibodies as well as Envto these receptors was readily detected, demonstrating that theincorporated proteins retained their native structures. The equilibriumdissociation constant for the interaction between an Env derived fromthe prototype HIV-1 strain IIIB for CXCR4 was approximately 500 nM,explaining the difficulty in measuring this interaction using standardequilibrium binding techniques. Retroviral pseudotypes represent easilyproduced, stable, homogenous structures that can be used to present awide array of single and multiple membrane-spanning proteins in a nativelipid environment for biosensor studies, thus avoiding the need fordetergent solubilization, purification, and reconstitution. The approachshould have general applicability and can be used to correlateEnv-receptor binding constants to viral tropism and pathogenesis.

[0290] The data disclosed herein demonstrate the development of a noveltechnique to study ligand binding to both topologically simple andcomplex transmembrane proteins using the optical biosensor by presentingthese proteins on the surface of retroviral particles. The datadisclosed herein demonstrate that a number of single-spanning proteinsand 7TM domain chemokine receptors can be incorporated into virions,which can be easily purified and attached to the biosensor surface.Binding of antibodies and HIV-1 gp120 to these receptors exhibitedappropriate specificity, and structural integrity of the receptors wasmaintained. Binding of a small-molecule inhibitor (ALX40-4C) wasdemonstrated by virtue of its ability to inhibit gp 120 binding. The useof these retroviral pseudotypes in the optical biosensor eliminates theneed to purify and reconstitute membrane proteins for ligand bindingstudies and provides a general experimental technique to characterizefunctionally important interactions with membrane proteins that wouldotherwise not be possible with standard equilibrium binding assays.

[0291] The materials and methods of this Example are as follows.

[0292] Proteins

[0293] HIV-1 HXBc2 and 8x gp120 were produced and purified by lectinchromatography (Hoffinan et al., 1999, Proc. Natl. Acad. Sci. USA96:6359-6364). The anti-gp120 mAb 17b was provided by J. Robinson(Tulane University, New Orleans) (Thali et al., 1993, J. Virol.67:3978-3988 and Kwong et al., 1998, Nature (London) 393:648-659). mAbsCTC8 and #549 to CCR5 and mAbs R&D#8 and R&D#16 were provided by M.Tsang (R & D Systems) (Lee et al., 1999, J. Biol. Chem. 274:9617--9626).mAbs 4G10 and 7C11.1 to CXCR4 were a gift of C. Broder (UniformedServices University of the Health Sciences, Bethesda) (Chabot et al.,2000 J. Virol. 74:4404-4413), anti-CXCR4 mAb 12G5 was described inEndres et al. (1996, Cell 87:745-756), and anti-CCR5 mAb 2D7 was fromResearch Diagnostics (Flanders, N.J.). ALX40-4C, a specific peptideinhibitor of CXCR4, was provided by Allelix (Salt Lake City). (Doranz etal., 1997, J. Exp. Med. 186:1395-1400). The murine antibody 9E10 wasused for detection of the myc epitope (Evan et al., 1985, Mol. Cell.Biol. 5:3610-3616). Chick collapsin-1 containing a histidine tail waspurified via nickel column chromatography (Koppel et al., 1997, Neuron19:531-537).

[0294] Pseudotype Production, Purification, and Characterization

[0295] Murine leukemia virus (MLV) pseudotypes were produced by calciumphosphate-mediated transfection of 293T cells in 225-cm 2 flasks with a3:1 ratio of receptor plasmid to pCGP, which encodes the MLV gag and polgenes. Four hours posttransfection, fresh media supplemented with 10 mMn-butyric acid was added to increase protein expression. 48 hoursposttransfection, supernatant was harvested, and cell debris was removedby low speed centrifugation and 0.45 μm filtration. The supernatant waspelleted for 90 minutes in an SW28 rotor at 28,000 RPM through 20%sucrose/PBS and resuspended overnight in PBS. A secondultracentrifugation step through 20% sucrose/PBS was per-formed in anSW40 rotor at 40,000 RPM for 45 minutes, and the pellet was resuspendedin 100 μl of 10 mM Hepes, pH 7.4. The pseudotypes were either stored at4° C. or aliquoted and frozen at −20° C. MLV pseudotypes were analyzedfor MLV gag and receptor expression by SDS-PAGE and Western blot.Pseudotypes were also analyzed by equilibrium density gradientultracentrifugation using a 1545% sucrose gradient at 35,000 RPM for 16hours in a SW40 rotor. Particles were also examined by negative stainelectron microscopy on carbon films after staining with uranyl acetate.

[0296] Attachment of Lipoparticles to Biosensor Surfaces

[0297] All attachments were performed in PBS running buffer usingBia2000 or BiaX optical biosensors (Biacore, Uppsala, Sweden) at 25° C.Lipoparticles, also referred to herein as “pseudotypes,” were attachedto a gold surface derivatized with a carboxylated alkane thiol (BiacoreC1 chip) or a short carboxy-dextran matrix (Biacore F1 chip) following a10-min activation of surface carboxyl groups using a 1:1 mixture of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (1 M)and N-hydroxysuccinimide (0.25 M) at 5 μl/min. Pseudotypes that had beenmixed 1:1 with 0.1 M sodium acetate, pH 5.5, were injected manuallyuntil the desired level of response units (RU), usually between 2,000and 6,000 RU, had been reached. Following attachment, the remainingsurface carboxyl groups were quenched with 35 μl of 1 M ethanolamine, pH8.5, at 5 μl/min.

[0298] Binding Experiments

[0299] Binding experiments were performed in DMEM with 0.1% PluronicF127 (Sigma) or PBS without surfactants at 30 μl/min and at 25° C.unless otherwise noted. Importantly, every binding experiment performedincluded a reference surface containing an equivalent RU amount of MLVparticles made with pCDNA3 or other receptor as a negative control.Analyte was removed following each binding interaction using duplicate20-μl pulses of regeneration solution at 100 μl/min. Regenerationconditions varied for each ligand analyte pair and were optimizedempirically to remove all bound protein and maintain surface activityusing various combinations of pH 5 (0.15 M oxalic acid/0.15 MH₃PO₄/0.15M formic acid/0.15 M malonic acid, pH 5), pH 9 (0.2 M ethanolamine/0.2 MNa₃PO₄/0.2 M glycine/0.2 M piperazine, pH 9), 1 M NaCl, 1 M MgCl₂, andchaotropic (0.46 M KCSN/1.83 M MgCl₂/0.92 M urea/1.83 M guanidine HCl)solutions (Andersson et al., 1999, Anal. Chem. 71:2475-2481). Dataanalysis and fitting was performed with BIAEVALUATION 3.0 software.

[0300] The Results of the experiments disclosed in this example are asfollows.

[0301] Receptor incorporation and Characterization of MLV Lipoparticles

[0302] Retroviruses can nonspecifically incorporate cell-surfacemembrane proteins into their lipid envelope as they bud from the plasmamembrane (Young et al., 1990, Science 250:1421-1423, Balliet et al.,1998, J. Virol. 72:671-676 and Suomalainen et al., 1994, J. Virol.68:4879-4889). To determine whether retroviral pseudotypes could be usedto present membrane proteins in their native conformations for opticalbiosensor studies, different transmembrane proteins were transientlycoexpressed with the structural proteins necessary to generate MLVparticles in 293T cells. The media were collected, and virus particleswere purified by ultracentrifugation and analyzed for the presence ofthe viral core protein (gag) and the desired membrane protein. The typeI membrane protein CD4 and the 7TM chemokine receptors CCR5 and CXCR4were incorporated into virus particles at readily detectable levels(FIG. 8A). The virus particles were judged pure by equilibrium gradientcentrifugation (FIG. 8B) and negative stain electron microscopy, whichrevealed a homogenous population of vesicular structures with an averagediameter of 105±29 nm.

[0303] Immobilization of MLV Lipoparticles to Biosensor Surfaces

[0304] To perform binding studies using the purified receptor-bearingMLV lipoparticles, virions were captured on a derivatized gold surfacesuitable for use in a Biacore optical biosensor. A number of sensorsurfaces are available from Biacore, each with different surfaceproperties. A standard coupling chemistry technique was used in whichsensor surface carboxyl groups are activated withN-hydroxysuccinimide/EDC, permitting subsequent formation of covalentbonds with primary amines on the virion surface. The most frequentlyused sensor chip (CM5) contains a ;100 nM dextran hydrogel derivatizedwith carboxyl groups (Myszka et al., 1999 J. Mol. Recognit. 12:390-408).

[0305] Because viral particles are likely to be negatively charged andsurface plasmon resonance decays exponentially as a function of distancefrom the biosensor surface, a carboxy-methylated surface that lacks adextran matrix (Biacore C1 chip) was used. About 4,000-6,000 RU of virusparticles were reliably attached to the C1 chip but it was difficult toobtain robust attachment of MLV particles onto the CM5 surface. However,suitable attachment (4,000-6,000 RU) could be obtained on a sensor chipwith a shorter dextran surface (Biacore F1 chip). The optimal pH forattachment of the pseudotypes was 5.5 for all surfaces and receptors.Following attachment, reductions in baseline were not observed with timeor repeated regeneration, indicating that the particles wereirreversibly linked to the sensor surface.

[0306] Antibody Binding Studies to MLV Lipoparticles ContainingChemokine Receptors.

[0307] Equivalent amounts of MLV particles containing CCR5 (MLV-CCR5),CXCR4 (MLV-CXCR4), or no receptor (MLV-pCDNA3) were attached to thebiosensor surface, and binding of specific antibodies was measured. Theanti-CXCR4 antibody 12G5, which recognizes a conformational epitope inCXCR4 (Doranz et al., 1999, J. Virol. 73:2752-2761 and Brelot et al.,1997, J. Virol. 71:4744-4751), bound to MLV-CXCR4 and did not bind toMLV-CCR5 or MLV-pCDNA3 (FIG. 9A). The anti-CCR5 antibody CTC8, whichrecognizes a linear epitope on the N terminus of CCR5 (Lee et al., 1999,J. Biol. Chem. 274:9617-9626), bound to MLV-CCR5 and did not bind toMLV-pCDNA3 or MLV-CXCR4 (FIG. 9B). When PBS was washed across the sensorsurface following injection of the antibodies, a typical dissociationcurve was observed (arrows in FIG. 9A and B). Similar results wereobtained with CCR5 mAbs #549 and 2D7 and CXCR4 mAbs 4G10, R&D#8, andR&D#16 to both linear and conformational epitopes. Mouse IgG and BSAshowed minimal binding to any of the MLV particles on the F1 or C1chips.

[0308] For retroviral lipoparticles to be successful vehicles forpresenting membrane proteins on a biosensor surface, they shouldwithstand multiple regeneration cycles in which bound analytes areremoved without damaging either the particles or the receptors theycontain. In this way, multiple binding experiments can be performed witha single surface, a prerequisite for the accurate determination ofbinding constants. The data disclosed herein demonstrate that a briefpulse with a regeneration mixture containing an equal proportion of pH 5and chaotropic solutions (Andersson et al., 1999, Anal. Chem.71:2475-2481) efficiently removed 12G5 from MLV-CXCR4 particles,returning the signal to baseline (FIG. 9A, bars). A single injection ofthis regeneration buffer was also sufficient to remove CTC8 fromMLV-CCR5 particles, again returning the signal to baseline (FIG. 9B,bar). Similar results were obtained with other CCR5 and CXCR4antibodies.

[0309] The reproducibility and stability of the MLV particles tomultiple binding and regeneration cycles is shown in FIG. 9C. Overlayplots from six sequential binding reactions on the same biosensorsurface performed with 12G5 were virtually identical. Results with CTC8were similar. These results indicate that the regeneration conditionsremoved antibody from the surface without damaging the MLV particles oraltering receptor conformation. In fact, binding experiments wereperformed over the course of several days before significant decreasesin the binding capacity of the MLV particles was observed on a givensensor chip. In addition, MLV particles were stored at −20° C. for atleast several weeks before attachment and use in biosensor experiments.

[0310] As an additional specificity control, the ability of ALX40-4C, asmall peptide inhibitor of CXCR4, to block 12G5 binding to MLV-CXCR4particles (Doranz et al., 1997, J. Exp. Med. 186:1395-1400), wasassessed. As shown in FIG. 9D, inclusion of ALX40-4C in the runningbuffer eliminated 12G5 binding to MLV-CXCR4 at a concentration (4 mM)similar to that needed to inhibit HIV-1 infection (Doranz et al., 1997,J. Exp. Med. 186:1395-1400). Furthermore, ALX40-4C could be washed outand full binding of 12G5 to MLV-CXCR4 restored (FIG. 9D). The ability ofCTC8 to bind MLV-CCR5 was unaffected by the presence of ALX40-4C. Thereversible ability of ALX40-4C to specifically prevent 12G5 binding toMLV-CXCR4 confirms the specificity of the lipoparticle system and alsoshows that this approach can be used to monitor binding of smallmolecules to membrane incorporated receptors.

[0311] Having shown that antibody binding to chemokine receptors on MLVpseudotypes was specific and highly reproducible, a series ofexperiments was performed to assess the kinetic constants of theseinteractions. Binding of 12G5 to CXCR4 particles at different flow ratesensured that ligand binding to the MLV particles was notdiffusion-limited (FIG. 9E). Results were similar for CTC8 and otheranti-CCR5 and anti-CXCR4 anti-bodies. Next, the binding of the mAbs CTC8and 12G5 to the chemokine receptors CCR5 and CXCR4 was measured using arange of antibody concentrations (FIGS. 9F and 3), and the data wereanalyzed using BIAEVALUATION 3.0 software. Analysis of the bindingcurves for 12G5 indicated that the data were consistent with a bivalentinteraction (X² 3.0 for 12G5 using the bivalent model with R_(max) 88)but not with a 1:1 interaction (X² 36 for the same data analyzed by the1:1 model).

[0312] These results are consistent with each 12G5 antibody binding twoCXCR4 receptors on the MLV particle. Similar fitting results wereobtained with CTC8 (FIG. 2F) and other anti-CCR5 and anti-CXCR4antibodies. Because antibody binding to the chemokine receptors wasbivalent, this will result in a higher apparent affinity, and thekinetics cannot be described with a simple interaction model. Thus, toaccurately measure antibody-receptor binding constants using thistechnique, Fab fragments can be used (Myszka et al., 1999, J. Mol.Recognit. 12:279-284).

[0313] Binding Studies of HIV-1 gp120 to MLV Pseudotypes.

[0314] Direct binding of X4 gp120 proteins to CXCR4 has been difficultto measure (Doranz et al., 1999, J. Virol. 73:2752-2761). In addition,without wishing to be bound by any particular theory and althoughbinding of gp120 subunits derived from R5X4 virus strains to CD4 can beeasily detected, binding of these proteins to CCR5, CXCR4, or othercoreceptors cannot, perhaps due to low affinity interactions (Doranz etal., 1999, J. Virol. 73:2752-2761, Baik et al., 1999, Virology259:267-273 and Etemad-Moghadam et al., 2000, J. Virol. 74:4433-4440).It was reasoned that the real-time nature of the biosensor would make itpossible to measure gp120-CXCR4 interactions more readily thantraditional binding methods that rely on steady-state measurements. Tosimplify the binding interaction, a gp120 from a CD4-independent strainof HIV-1 termed 8x, which interacts directly with CXCR4 (Hoffinan etal., 1999, Proc. Natl. Acad. Sci. USA 96:6359-6364), was used.

[0315] Initial attempts to measure specific 8x gp120 binding toMLV-CXCR4 on a C1 chip in PBS running buffer were unsuccessful due tononspecific binding associated with this highly glycosylated protein tothe control surface. When the running buffer was changed from PBS toDMEM with 0.1% Pluronic F127, a surfactant previously shown to decreasethe nonspecific binding of proteins to gold surfaces (Green et al.,1998, J. Biomed. Mater. Res. 42:165-171), specific binding of 8x toMLV-CXCR4 compared with MLV-pCDNA3 was detected (FIG. 11A). Binding of8x could be prevented by the 17b antibody, which binds to the coreceptorbinding site in gp120 (Kwong et al., 1998, Nature 393:648-659),confirming the specificity of this interaction (FIG. 12B). Similarresults were obtained when binding studies were performed with 8x in PBSrunning buffer without Pluronic F127 on an F1 chip, which reduced, butdid not completely eliminate, the nonspecific binding of Env.

[0316] Regeneration conditions (pH 5/chaotropic solution) used to remove12G5 and other anti-CXCR4 antibodies also proved to be successful forstripping gp120 from MLV-CXCR4 (FIG. 11A). Multiple binding andregeneration cycles of 8x indicated that there was approximately a 2%loss in subtracted signal with each Env binding interaction. Withoutwishing to be bound by any particular theory, this may be related toirreversible removal of lipoparticles from the surface such as by lysisof the lipid membrane, inactivation of CXCR4 conformations involved inEnv binding, or incomplete removal of Env from the sensor surfacefollowing regeneration.

[0317] When dose-response experiments with 8x gp120 were performed (FIG.11C), the equilibrium dissociation constant was calculated to be 506 ±nM (from five independent experiments). The fast off-rate exhibited by8x gp120 helps explain the difficulty experienced in measuring thisinteraction using standard equilibrium cell-surface binding assays, asall or most of the gp120 dissociates from CXCR4 by the time the washingsteps are complete. Finally, the data disclosed herein demonstrate thatspecific binding of a CD4-dependent gp120 from the HXB strain of HIV-1to MLV-CXCR4 was detected, but only when soluble CD4 was included in therunning buffer so as to trigger the conformational changes in gp 120needed for coreceptor binding, further demonstrating that thelipoparticle construct preserves the biological structure and functionof the protein embedded in the lipid bilayer of the construct.

[0318] Binding Studies with Other Membrane Proteins.

[0319] Having established retroviral pseudotyping as a way to constructlipoparticles in order to study binding interactions with a biosensor,we determined whether binding to other membrane proteins could bemeasured with this technique. Neuropilin-(NP-1) is a member of a relatedgroup of type 1 membrane proteins involved in axonal guidance in thedeveloping nervous system. A family of protein ligands, termedcollapsing, binds to NP-1 receptors on axons and triggers axonalrepulsion and redirection (He et al., 1997, Cell 90:739-751). Afterdetermining that NP-1 and a similar protein, plexin-2, could beincorporated into MLV particles (FIG. 12A), we attached MLV-NP-1 to abiosensor surface and measured binding of collapsin-1. As shown in FIG.12B, collapsing specifically interacted with MLV-NP-1. These resultsindicate that a diverse group of membrane proteins can be incorporatedand presented in MLV particles for binding studies in the opticalbiosensor.

[0320] Optical biosensor technology can be used to study molecularinteractions in real time, making it possible to accurately measurekinetic and equilibrium binding constants (Canziani et al., 1999,Methods 19:253-269 and Rich et al., 2000, Curr. Opin. Biotechnol.11:54-61). Whereas interactions between soluble molecules can beroutinely measured, it has not been possible to present membraneproteins in their native, lipid environments on the sensor surface for anumber of technical reasons. These problems can sometimes becircumvented by generating soluble ectodomain fragments of type I andtype II integral membrane proteins, but proteins that span the membranemultiple times or that exist as multimeric complexes are not as easilymanipulated.

[0321] In principle, integral membrane proteins can be purified andreconstituted into artificial membranes that can be attached to thesensor surface. However, purification and reconstitution of membraneproteins is a laborious and empirically driven process, and thus far ithas not been used successfully in an optical biosensor format with theexception of bacterial rhodopsin (Salamon et al., 1994, Biochemistry33:13706-13711). As a result, entire classes of membrane proteins, suchas seven transmembrane domain receptors, have not been studied usingthis technique. In the case of HIV, there are many instances in whichstandard equilibrium binding assays are not sufficiently sensitive tostudy in detail, or sometimes even detect, interactions between theviral Env protein and its 7TM coreceptors (Doranz et al., 1999, J.Virol. 73:2752-2761, Baik et al., 1999, Virology 259:267-273 andEtemad-Moghadam et al., 2000, J. Virol. 74:4433-4440).

[0322] Because HIV-coreceptor interactions are critically importantdeterminants of viral tropism and pathogenesis, and because thesereceptors are important drug targets (Berger et al., 1999, Annu. Rev.Immunol. 17:657-700), this is a significant shortcoming. Therefore,lipoparticles were produced by taking advantage of the fact thatretroviruses can incorporate cellular membrane proteins into their lipidenvelopes during the process of budding from the cell surface. Ineffect, retroviral pseudotypes serve as model membrane vesicles that,due to the presence of the viral core, are homogeneous in size, easilypurified, and stable. In addition, incorporation of a membrane proteininto a retrovirus avoids the need for detergent solubilization,purification, and reconstitution. A significant number of cellularmembrane proteins can be incorporated into retroviral pseudotypes,indicating that the approach described here should be broadlyapplicable.

[0323] A host of type I, type II, and multiple membrane-spanningcellular membrane proteins have been previously shown to be incorporatedinto retrovirus particles, including class I and class II MHC proteins,CD4, various ICAMs, a tetraspan protein (CD63), as well as multiplemembrane-spanning proteins such as the murine cationic amino acidtransporter, which functions as a receptor for the ecotropic murineleukemia virus (see, e.g., Balliet & Bates, 1998, J. Virol. 72:671-676,and references cited therein). For this approach to work in the opticalbiosensor format, the incorporated membrane proteins must retain theirnative conformation as has been demonstrated using the lipoparticlesdisclosed herein.

[0324] Studies in which viral receptors are incorporated into retroviralparticles, enabling these particles to infect cells expressing thecognate viral Env glycoproteins, demonstrate this. For example,incorporation of CD4 and either CCR5 or CXCR4 into virus particlesenables these virions to infect cells expressing R5 or X4 HIV-1 Envproteins, respectively (Endres et al., 1997, Science 278:1462-1464 andSchnell et al., 1997, Cell 90:849-857). Because the determinants on CD4and CCR5 recognized by the viral Env protein are conformationallycomplex (Hoffinan et al., 1998, AIDS 12, Suppl. A, S17-S26), theseresults indicate that the pseudotyped receptors retain their nativeconformation. In addition, this shows that at least two differentproteins can be incorporated into a given virus particle (e.g., heteroor homo-oligomer complexes); it also shows that because membrane fusionis a cooperative process requiring multiple receptor binding events(Hernandez et al., 1996, Annu. Rev. Cell Dev. Biol. 12:627-661),multiple copies of each can be incorporated. In the case of HIV-1, it isestimated that six CCR5 molecules are needed to support membrane fusion(Kuhmann et al., 2000 J. Virol. 74:7005-7015) and that multiple CD4molecules are also needed (Layne et al., 1990, Nature 346:277-279). Thedata disclosed herein support these conclusions in that the 7TM and typeI membrane proteins studied herein retained their native conformationsas judged by their abilities to bind a variety of conformationallysensitive ligands. The presence of bivalent interactions also suggeststhat there is lateral mobility in the retroviral membrane, providingfurther evidence that they are a good cell-surface surrogate.

[0325] The efficiency with which a protein can be pseudotyped into avirus particle can be influenced by the location and degree ofexpression and the nature of the cytoplasmic domain of the protein(Young et al., 1990, Science 250:1421-1423; Suomalainen et al., 1994, J.Virol. 68:4879-4889; Suomalainen et al., 1994, J. Virol. 68:4879-4889;and Henriksson et al., 2000, J. Virol. 73:9294-9302). A prerequisite forpseudotype formation with MLV is that the protein of interest beexpressed on the cell membrane from which the virus buds. Potentially,viruses that bud from intracellular compartments can be used toincorporate cellular membranes that reside elsewhere in the cell.Alternatively, proteins retained in intracellular organelles can beretargeted to the cell surface and incorporated into, for instance, MLVparticles by modifying retention or targeting motifs.

[0326] There is increasing evidence that some viruses selectively budfrom the cell surface through detergent-insoluble lipid rafts (Nguyen etal., 2000, J. Virol. 74:3264-3272; Zhang et al., 2000, J. Virol.74:4634-4644). Therefore, targeting proteins of interest to lipid raftscould, for some virus types, improve pseudotype formation. Once theprotein is expressed at the proper location on the cell surface,incorporation efficiency is likely to be related to expression levels.Because high level expression is desirable, a transient expressionsystem using a cell type that is easily transfectable as well as beingcapable of high levels of protein production was used. In excess of100,000 CCR5 and CXCR4 molecules are expressed per cell using thisapproach (Lee et al., 1999, J. Biol. Chem. 274:9617-9626).

[0327] Lipoparticle formation can be improved by constructing chimericmolecules in which the cytoplasmic domain of a membrane protein isreplaced with that of the retroviral Env protein, or any tag that linksthe surface molecule to the structural proteins of the virus (gag).Shortening a long cytoplasmic region can also improve proteinincorporation into viral pseudotypes by reducing negative interactionsbetween bulky cytoplasmic domains and retroviral gag protein (Henrikssonet al., 2000, J. Virol. 73:9294-9302). A final factor to consider is thetype of virus used. In addition to MLV, other viruses such as vesicularstomatitis virus, RSV, rabies viruses, and HIV can also be used togenerate pseudotypes, providing additional options for packagingcellular membrane proteins into virus particles (Endres et al., 1997,Science 278:1462-1464 and Schnell et al., 1997, Cell 90:849-857).

[0328] The use of retroviral pseudotypes as membrane presentationvehicles, i.e., lipoparticles, will make it possible to study ligandinteractions with many different cellular membrane proteins usingmicrofluidic devices in general and optical biosensors in particular.This approach has important implications for drug discovery in whichbinding of small molecules to 7TM and other membrane receptors can bemeasured. The data disclosed herein demonstrate attachment of 4,000 to6,000 RUs of virus particles allowed several hundred RUs of specificantibody binding to be obtained. This is considerably in excess of whatis needed to obtain accurate kinetic measurements, as accurate responsescan be measured well below 100 RU, and even below 10 RU in some cases(Myszka et al., 1999, J. Mol. Recognit. 12:279-284). Thus, the number oflipoparticles that should be bound to the sensor surface can be readilyassessed and modified as required pursuant to the teachings providedherein or as would be understood by the skilled artisan armed with theteachings of the invention.

[0329] It has been demonstrated that it is possible to detect binding oflow mass compounds using an optical biosensor (Markgren et al., 1999,Anal. Biochem. 265:340 350 and Strandh et al., 1998, J. Mol. Recognit.11:188-190). Because the signal measured by the optical biosensor isproportional to mass, it is likely that improved attachment ofretroviral pseudotypes will be needed to measure binding of smallmolecular weight compounds. Attachment of larger amounts of pseudotypesshould be possible because the data disclosed herein demonstrates thatthe binding capacity of the surfaces used herein was low so as tominimize mass transport effects that could be associated with the highmolecular weight ligands that were used herein (Myszka et al., 1999, J.Mol. Recognit. 12:279-284). It will be apparent to one skilled in theart based upon the disclosure provided herein, that an increase insignal (RU) can be readily obtained by increasing the number ofreceptors on each lipoparticle and/or by increasing the number oflipoparticles on the surface of a biosensor.

[0330] The ability to measure binding of small molecules to membranereceptors with an optical biosensor could make this a useful screeningtool. Advantages of this approach include the fact that only a smallamount of sample is needed and that the ligand does not have to belabeled. The ability of many compounds to bind a given receptor could berapidly screened, making it possible to identify compounds withdesirable association and dissociation kinetics, information notnormally available from other screening methods. In the case of HIV-1,this approach should make it possible to directly measure someEnv-receptor interactions, providing information on the relationshipbetween Env-receptor affinity and viral tropism and pathogenesis, andalso on how small molecule inhibitors interact with the major HIVcoreceptors and block Env binding and viral infection.

[0331] The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

[0332] While this invention has been disclosed with reference tospecific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed is:
 1. An isolated lipoparticle comprising a multiplemembrane spanning protein wherein said protein is not CD63.
 2. Thelipoparticle of claim 1, wherein said protein is capable of binding witha ligand under conditions wherein said ligand would bind with anotherwise identical protein present on a cell membrane.
 3. The isolatedlipoparticle of claim 1, wherein said lipoparticle is a virus.
 4. Theisolated lipoparticle of claim 3, wherein said virus is amembrane-enveloped virus.
 5. The isolated lipoparticle of claim 4,wherein said membrane-enveloped virus is a retrovirus.
 6. The isolatedlipoparticle of claim 4, wherein said virus is selected from the groupconsisting of a murine leukemia virus, a human immunodeficiency virus, arabies virus, a Rous sarcoma virus, and a vesicular stomatitis virus. 7.The isolated lipoparticle of claim 1, wherein said protein is selectedfrom the group consisting of a G-protein coupled receptor, a transporterprotein, and an ion channel protein.
 8. The isolated lipoparticle ofclaim 1, wherein said protein is selected from the group consisting ofCCR5, CXCR4,, MCAT-1, CXCR2, CXCR3, mu-opioid receptor, and KCNH2potassium channel protein.
 9. A composition comprising an isolatedlipoparticle attached to a sensor surface, said lipoparticle furthercomprising a membrane spanning protein.
 10. The composition of claim 9,wherein said protein is selected from the group consisting of atransport protein, a G-protein coupled receptor, an ion channel protein,a type I membrane protein, and a type II membrane protein.
 11. Thecomposition of claim 10, wherein said G-protein coupled receptor isselected from the group consisting of a mu-opioid receptor, a CXCR2,CXCR3, CXCR4, a CCR5, a CCR8, a XCR1, and a CX3CR1.
 12. The compositionof claim 10, wherein said ion channel protein is selected from the groupconsisting of KCNH2 potassium channel protein, Kv1.3 potassium channelprotein, and CFTR protein.
 13. The composition of claim 10, wherein saidtransporter protein is selected from a group consisting of a glucosetransporter protein and an amino acid transporter protein.
 14. Thecomposition of claim 10, wherein said type I membrane protein isselected from the group consisting of CD4, Tva, and neuropilin-2. 15.The composition of claim 10, wherein said type II membrane proteincomprises DC-specific ICAM-3 grabbing nonintegrin (DC-SIGN).
 16. Thecomposition of claim 9, wherein said lipoparticle is a virus.
 17. Thecomposition of claim 16, wherein said virus is a membrane-envelopedvirus.
 18. The composition of claim 17, wherein said membrane-envelopedvirus is a retrovirus.
 19. The composition of claim 17, wherein saidvirus is selected from the group consisting of a murine leukemia virus,a human immunodeficiency virus, a rabies virus, a Rous sarcoma virus,and a vesicular stomatitis virus.
 20. The composition of claim 9,wherein said lipoparticle further comprises a plastic bead core to forma proteoliposome.
 21. The composition of claim 9, wherein said sensorcomprises a microfluidic device.
 22. The composition of claim 21,wherein said microfluidic device is a biosensor.
 23. The composition ofclaim 22, wherein said biosensor is an optical biosensor.
 24. Thecomposition of claim 23, wherein said optical biosensor measures surfaceplasmon resonance (SPR).
 25. The composition of claim 23, wherein saidsurface is located on a biosensor chip.
 26. The composition of claim 25,wherein said biosensor chip is selected from the group consisting of agold coated biosensor chip, a gold and dextran coated biosensor chip,and a derivatized gold biosensor chip.
 27. A method of assessing thebinding interaction of a membrane spanning protein with a ligand, saidmethod comprising (a) producing a lipoparticle comprising a membranespanning protein; (b) attaching said lipoparticle to a substrate; (c)contacting said protein present on said lipoparticle with a ligand ofsaid protein; and (d) detecting any change in said substrate comparedwith any change in an otherwise identical substrate wherein said proteinpresent on said lipoparticle is not contacted with said ligand, whereindetecting a change in said substrate wherein said protein present onsaid lipoparticle is contacted with said ligand compared with saidotherwise identical substrate wherein said protein present on saidlipoparticle is not contacted with said ligand assesses said bindinginteraction of said protein with said ligand.
 28. The method of claim27, wherein said detecting in (d) is performed using a microfluidicdevice and said substrate is a sensor surface.
 29. The method of claim28, wherein said microfluidic device is a biosensor device.
 30. Themethod of claim 27, wherein said biosensor device comprises amicrochannel or a microwell.
 31. The method of claim 29, wherein saidbiosensor is an optical biosensor.
 32. The method of claim 31, whereinsaid optical biosensor is a surface plasmon resonance biosensor device.33. A method of identifying a potential ligand of a membrane protein,said method comprising (a) attaching a lipoparticle comprising amembrane protein to a surface; (b) contacting said protein with a testligand; and (c) comparing said surface comprising said lipoparticlecomprising said protein contacted with said test ligand with anotherwise identical surface comprising an otherwise identicallipoparticle comprising a protein not contacted with said test ligand,wherein a difference between said surface comprising said lipoparticlecomprising a protein contacted with said test ligand compared with saidotherwise identical surface comprising said otherwise identicallipoparticle comprising said protein not contacted with said test ligandis an indication that said ligand is a potential ligand of said protein.34. The method of claim 33, wherein said comparing in (c) is performedusing a microfluidic device.
 35. The method of claim 34, wherein saidmicrofluidic device is a biosensor device.
 36. The method of claim 33,wherein said protein is selected from a multiple membrane spanningprotein and a single membrane spanning protein.
 37. The method of claim33, wherein said multiple membrane spanning protein is selected from thegroup consisting of a G-coupled protein receptor (GCPR), a transporter,and an ion channel.
 38. The method of claim 36, wherein said singlemembrane spanning protein is selected from the group consisting of atype I membrane protein and a type II membrane protein.
 39. The methodof claim 33, wherein said test ligand is selected from the groupconsisting of a protein and a chemical compound.
 40. The method of claim39, wherein said protein is an antibody.
 41. A ligand identified by themethod of claim
 33. 42. A method of identifying a compound that affectsbinding between a ligand and a membrane protein receptor, said methodcomprising (a) attaching a lipoparticle comprising a membrane protein toa surface; (b) contacting said protein with a known ligand underconditions wherein said protein specifically binds with said ligand; (c)contacting said lipoparticle of (b) with a test compound; and (d)comparing said surface comprising said lipoparticle contacted with saidtest compound with an otherwise identical surface comprising anotherwise identical lipoparticle not contacted with said test compound,wherein a difference between said surface comprising said lipoparticlecontacted with said test compound compared with said otherwise identicalsurface comprising said otherwise identical lipoparticle not contactedwith said test compound is an indication that said test compound affectsbetween said ligand and said membrane protein receptor.
 43. A kit forassessing the binding interaction of a membrane spanning protein with aligand, said kit comprising a lipoparticle comprising a membranespanning protein and a substrate, said kit further comprising anapplicator, and an instructional material for the use thereof.
 44. Thekit of claim 43, said kit further comprising a ligand of said protein.45. A kit for identifying a potential ligand of a membrane protein, saidkit comprising a lipoparticle comprising a membrane protein and asurface, said kit further comprising an applicator, and an instructionalmaterial for the use thereof.
 46. The kit of claim 45, said kit furthercomprising a test ligand.
 47. A kit for identifying a compound thataffects binding between a ligand and a membrane protein receptor, saidkit comprising a lipoparticle comprising a membrane protein and asurface, said kit further comprising an applicator, and an instructionalmaterial for the use thereof.
 48. The kit of claim 47, said kit furthercomprising a test compound.
 49. The kit of claim 47, said kit furthercomprising a known ligand of said membrane protein.